Exogenous Co-Reactant-Free Electrochemiluminescent Biosensor for Ratiometric Measurement of α-Glucosidase Based on a ZIF-67-Regulated Hydrogen-Bonded Organic Framework.

The development of highly sensitive and selective analytical approaches for monitoring enzymatic activity is critical for disease diagnosis and biomedical research. Herein, we develop an exogenous co-reactant-free electrochemiluminescence (ECL) biosensor for the ratiometric measurement of α-glucosidase (α-Glu) based on a zeolitic imidazolate framework (ZIF-67)-regulated pyrene-based hydrogen-bonded organic framework (HOF-101). Target α-Glu can hydrolyze maltose to α-d-glucose, which can subsequently react with GOx to produce gluconic acid. The resultant gluconic acid can dissolve ZIF-67, leading to the recovery of the HOF-101 cathodic ECL signal and the decrease of the luminol anodic ECL signal. The long-range ordered structure of HOF-101 can speed up charge transfer, resulting in a stable and strong cathodic ECL signal. Moreover, ZIF-67 can not only efficiently quench the ECL signal of HOF-101 due to ECL resonance energy transfer between HOF-101 and ZIF-67 as well as the steric hindrance effect of ZIF-67 but also enhance the anodic ECL emission of luminol in dissolved O2 system because of its ordered and porous crystalline structure and the atomically dispersed Co2+. Notably, HOF-101 possesses a higher ECL efficiency (32.22%) compared with the Ru(bpy)32+ standard. Importantly, this ratiometric ECL biosensor shows high sensitivity (a detection limit of 0.19 U L-1) and a broad linear range (0.2-50 U L-1). This biosensor can efficiently eliminate systematic errors and enhance detection reliability without the involvement of exogenous co-reactants, and it displays good assay performance in human serum samples, holding great promise in biomedical research studies.

Phase I clinical trial of EGFR-specific CAR-T cells generated by the piggyBac transposon system in advanced relapsed/refractory non-small cell lung cancer patients.

PURPOSE: This phase I clinical trial is designed to assess the safety and feasibility of the epidermal growth factor receptor (EGFR) chimeric antigen receptor (CAR) T-cell generated by the piggyBac transposon system in advanced relapsed/refractory non-small cell lung cancer (NSCLC) patients. Compared to viral systems, the piggyBac transposon system is a simpler, more economical, and alternative way to introduce chimeric antigen receptor (CAR) transgenes into T cells. METHODS: This study recruited nine patients with advanced relapsed/refractory EGFR-positive NSCLC for two cycles of the piggyBac-generated EGFR-CAR T cells at dose of 1 × 106 cells/kg or 3 × 106 cells/kg of body weight. The patients were monitored for adverse events, clinical response, and persistence of plasma GFR-CAR T cells. RESULTS: Infusions of piggyBac-generated EGFR-CAR T cells were well tolerated in all nine patients. The most common adverse events were grade 1 to 3 fever and there were no patients who experienced grade 4 adverse events or serious cytokine release syndrome. After treatment, eight of nine patients showed detectable EGFR-CAR T cells in their peripheral blood. One patient showed a partial response and lasted for more than 13 months, while six had stable disease, and two had progressed disease. The progression-free survival of these nine patients was 7.13 months (95% CI 2.71-17.10 months), while the median overall survival was 15.63 months (95% CI 8.82-22.03 months). CONCLUSION: This Phase I clinical trial revealed that the non-viral piggyBac transposon system-engineered EGFR-CAR T-cell therapy is feasible and safe in treatment of EGFR-positive advanced relapsed/refractory NSCLC patients. Future study will assess it in more patients or even possibly with a higher dose. Trial registration number NCT03182816.

αPD-1-mesoCAR-T cells partially inhibit the growth of advanced/refractory ovarian cancer in a patient along with daily apatinib.

Epithelial ovarian cancer (EOC) is the leading cause of death among gynecological malignancies in China. In particular, advanced/refractory ovarian cancer lacks effective targeted therapies due to the immunosuppressive and proangiogenic tumor microenvironment. Mesothelin (MSLN) has been found to be highly expressive in most EOC. Targeting MSLN by antibodies or chimeric antigen receptor-modified T (CAR-T) cells and immune checkpoint blockades as well as apatinib, an anti-angiogenic drug, have been used in patients with refractory ovarian cancer. Apatinib was reported to promote the infiltration of CD8+ T cells in lung cancer. However, the combination therapy of CAR-T secreting anti-PD-1 antibody with apatinib in EOC has not been reported. CASE PRESENTATION: Here we report a case of refractory EOC in a patient who had relapsed after multiline chemotherapy. The patient received autologous T cells that contained sequences encoding single-chain variable fragments specific for MSLN and full-length antibody for PD-1 (αPD-1). The modified T cells were called αPD-1-mesoCAR-T cells. After infusion, the copy number and PD-1 antibody secretion of the CAR-T cells were increased in the blood. By application of multimodality tumor tracking, MRI of the liver showed shrinkage of metastatic nodules from average diameter of 71.3-39.1 mm at month 2. The patient achieved partial response and survived more than 17 months. IL-6 levels in the patient fluctuated from the baseline to 2-4-folds after treatment, but side effects were mild with only grade 1 hypertension and fatigue. CONCLUSION: αPD-1-mesoCAR-T cell therapy combined with apatinib demonstrates a potential therapeutic effect on advanced refractory ovarian cancer. TRIAL REGISTRATION NUMBER: NCT03615313.

Bi-specific ligand-controlled chimeric antigen receptor T-cell therapy for non-small cell lung cancer.

Our goal is to develop a switch-controlled approach to enable better control of reactivity and safety of chimeric antigen receptor (CAR)-T therapy for non-small-cell lung cancer (NSCLC). Lentiviral transduction was performed to generate anti-FITC CAR-T cells and target cells stably expressing either isoform of the folate receptor. Colorimetric-based cytotoxic assay, enzyme-linked immunosorbent assay, and multiparametric flow cytometry analysis were used to evaluate the specificity and activity of CAR-T cells in vitro. Human primary T cells stably expressing the fully human anti-FITC CAR were generated. Anti-FITC CAR-T cells displayed antigen-specific and folate-FTIC dependent reactivity against engineered A549-FRα and THP-1-FRß. The selective activation and proliferation of anti-FITC CAR-T cells in vitro stringently relied on the co-existence of folate-FITC and FR- expressing target cells and was dose-titratable with the folate-FITC switch. The excellent in vitro efficacy and specificity of an adaptor-controlled CAR-T therapy to target both tumor cells and tumor-associated macrophages in NSCLCs were validated.

Enhanced human enterovirus 71 infection by endocytosis inhibitors reveals multiple entry pathways by enterovirus causing hand-foot-and-mouth diseases.

BACKGROUND: Human enterovirus 71 (EV71) was previously known to enter cells through clathrin or caveolar mediated endocytic pathways. However, we observed chlorpromazine (CPZ) or dynasore (DNS), which inhibit clathrin and dynamin mediated endocytosis, did not suppress EV71 cell entry in particular cell types. So the current knowledge of entry mechanisms by EV71 is not complete. METHODS: Viral infection was examined by flow cytometry or end-point dilution assays. Viral entry was monitored by immunofluorescence or pseudoviral infections. Various inhibitors were utilized for manipulating endocytic pathways. Cellular proteins were knockdown by siRNA. RESULTS: CPZ and DNS did not inhibit but rather enhance viral infection in A549 cells, while they inhibited infections in other cells tested. We further found CPZ did not affect EV71 binding to target cells and failed to affect viral translation and replication, but enhanced viral entry in A549 cells. Immunofluorescence microscopy further confirmed this increased entry. Using siRNA experiment, we found that the enhancement of EV71 infection by CPZ did not require the components of clathrin mediated endocytosis. Finally, CPZ also enhanced infection by Coxackivirus A16 in A549 cells. CONCLUSIONS: CPZ and DNS, previously reported as EV71 entry inhibitors, may rather lead to increased viral infection in particular cell types. CPZ and DNS increased viral entry and not other steps of viral life cycles. Therefore, our study indicated an unknown dynamin-independent entry pathway utilized by enteroviruses that cause Hand-Foot-and-Mouth Diseases.

Common genetic heterogeneity of human interleukin-37 leads to functional variance.

Interleukin-37 (IL-37) is an inhibitory member of the IL-1 family of cytokines. We previously found that balanced selection maintains common variations of the human IL37 gene. However, the functional consequences of this selection have yet to be validated. Here, using cells expressing exogenous IL-37 variants, including IL-37 Ref and IL-37 Var1 and Var2, we found that the three variants of IL-37 exhibited different immunoregulatory potencies in response to immune stimulation. The protein level of IL-37 Var2 was found to be significantly less than that of IL-37 Ref or Var1, despite the comparable mRNA levels of all three variants. Further study showed that IL-37 Var2 was rapidly degraded by a proteasome-dependent mechanism mediated by enhanced polyubiquitination, leading to a transient upregulation of IL-37 Var2 after immune stimulation. Finally, when ectopically expressed in cells, human IL-37 Var2 exerted less inhibition on proinflammatory cytokine production than did other IL-37 variants. Conversely, purified extracellular IL-37 variant proteins demonstrated comparable inhibitory abilities in vitro. In conclusion, our study reveals that common genetic variants of IL37 lead to different immune-inhibitory potencies, primarily as a result of differences in IL-37 protein stability, suggesting the possible involvement of these variants in various human diseases.