Current Status and Perspectives of Dual-Targeting Chimeric Antigen Receptor T-Cell Therapy for the Treatment of Hematological Malignancies.

Single-targeted chimeric antigen receptor (CAR) T cells tremendously improve outcomes for patients with relapsed/refractory hematological malignancies and are considered a breakthrough therapy. However, over half of treated patients experience relapse or refractory disease, with antigen escape being one of the main contributing mechanisms. Dual-targeting CAR T-cell therapy is being developed to minimize the risk of relapse or refractory disease. Preclinical and clinical data on five categories of dual-targeting CAR T-cell therapies and approximately fifty studies were summarized to offer insights and support the development of dual-targeting CAR T-cell therapy for hematological malignancies. The clinical efficacy (durability and survival) is validated and the safety profiles of dual-targeting CAR T-cell therapy are acceptable, although there is still room for improvement in the bispecific CAR structure. It is one of the best approaches to optimize the bispecific CAR structure by boosting T-cell transduction efficiency and leveraging evidence from preclinical activity and clinical efficacy.

Detection and quantification of the age-related sjTREC decline in human peripheral blood.

The decline of signal joint T-cell receptor rearrangement excision circles (sjTRECs) in human peripheral blood has been demonstrated to be age-related, which can be a potential marker for individual age determination. However, little is known about the quantitative relationship between the levels of sjTREC and age. The aim of the present study was to investigate the levels of sjTREC in peripheral blood leukocytes (PBLs) among different age groups in Chinese population, so as to clarify whether it could serve as a suitable marker for biological age estimation in forensic practice. sjTREC levels were measured by real-time quantitative PCR analysis in peripheral blood samples from individuals of known age (n = 248). The quantification results showed that sjTREC declined in human PBLs in an age-dependent manner (r = -0.8177, P < 0.01). The formula for age estimation based on peripheral sjTREC decline was Y = -24.921x - 39.932 ± 10.47 (Y age, year; X log sjTREC/TBP; 10.47: standard error). Furthermore, there was no difference between males and females with regard to sjTREC levels. These results suggest that assessment of sjTREC in PBLs might be a valuable additional tool in age determination, especially in cases where traditional morphologic information is absent or inefficient in forensic practice.

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.

Pseudopterosin and O-Methyltylophorinidine Suppress Cell Growth in a 3D Spheroid Co-Culture Model of Pancreatic Ductal Adenocarcinoma.

Current therapies for treating pancreatic ductal adenocarcinoma (PDAC) are largely ineffective, with the desmoplastic environment established within these tumors being considered a central issue. We established a 3D spheroid co-culture in vitro model using a PDAC cell line (either PANC-1 or Capan-2), combined with stellate cells freshly isolated from pancreatic tumors (PSC) or hepatic lesions (HSC), and human type I collagen to analyze the efficiency of the chemotherapeutic gemcitabine (GEM) as well as two novel drug candidates derived from natural products: pseudopterosin (PsA-D) and O-methyltylophorinidine (TYLO). Traditional 2D in vitro testing of these agents for cytotoxicity on PANC-1 demonstrated IC50 values of 4.6 (±0.47) nM, 34.02 (±1.35) µM, and 1.99 (± 0.13) µM for Tylo, PsA-D, and GEM, respectively; these values were comparable for Capan-2: 5.58 (±1.74) nM, 33.94 (±1.02) µM, and 0.41 (±0.06) µM for Tylo, PsA-D, and GEM, respectively. Importantly, by assessing the extent of viable cells within 3D co-culture spheroids of PANC-1 with PSC or HSC, we could demonstrate a significant lack of efficacy for GEM, while TYLO remained active and PsA-D showed slightly reduced efficacy: GEM in PANC-1/PSC (IC50 = >100 µM) or PANC-1/HSC (IC50 = >100 µM) spheroids, TYLO in PANC-1/PSC (IC50 = 3.57 ± 1.30 nM) or PANC-1/HSC (IC50 = 6.39 ± 2.28 nM) spheroids, and to PsA-D in PANC-1/PSC (IC50 = 54.42 ± 12.79 µM) or PANC-1/HSC (IC50 = 51.75 ± 0.60 µM). Microscopic 3D rendering supported these cytotoxicity outcomes, showing little or no morphological spheroid structure change during this period of rapid cell death. Our results support the use of this 3D spheroid co-culture in vitro model having a desmoplastic microenvironment for the identification of possible novel chemotherapeutic drug candidates for PDAC, such as TYLO and PsA-D.

Expression of indoleamine 2,3-dioxygenase in nasopharyngeal carcinoma impairs the cytolytic function of peripheral blood lymphocytes.

BACKGROUND: Tumor-specific cytotoxic T cells and infiltrating lymphocytes are frequently found in tumor tissues in patients with nasopharyngeal carcinoma (NPC). Most patients with NPC, however, especially those with advanced stages, have a poor clinical prognosis despite conventional immunotherapy. The aim of this work was to examine the effect of indoleamine 2,3-dioxygenase (IDO), an immunosuppressive enzyme, on the lymphocyte function in NPC. METHODS: The NPC cell line CNE2 was treated by interferon-gamma (IFNgamma) and the levels of IDO expression was analyzed by Western blotting and reverse phase high-performance liquid chromatography (HPLC). Lymphocytes from health human exposed to the milieu created by IDO-positive CNE2 cells and the lymphocyte cytotoxicity to target tumor cells was analyzed by standard lactate dehydrogenase (LDH) release assay. Additionally, expression of IDO was determined by Immunohistochemical assay in the tumor tissues form clinically evaluated NPC. RESULTS: IDO expression was acutely induced in the NPC cell line CNE2 by low dose interferon-gamma (IFNgamma) or by co-incubation with activated lymphocytes. Exposure to the milieu created by IDO-positive CNE2 cells did not promote lymphocyte death, but lymphocyte cytotoxicity against target tumor cells was impaired. The suppression of lymphocyte cytotoxic function was fully restored when the conditioned medium was replaced by fresh medium for 24 h. In additionally, the IDO-positive cells were found scattered in the tumor tissues from patients with NPC. CONCLUSION: Altogether, these findings suggest that IDO-mediated immunosuppression may be involved in the tumor immune evasion, and that blocking IDO activity in tumor cells may help to re-establish an effective anti-tumor T cell response in NPC.

Intestinal P-glycoprotein exports endocannabinoids to prevent inflammation and maintain homeostasis.

Neutrophil influx into the intestinal lumen is a critical response to infectious agents, but is also associated with severe intestinal damage observed in idiopathic inflammatory bowel disease. The chemoattractant hepoxilin A3, an eicosanoid secreted from intestinal epithelial cells by the apically restricted efflux pump multidrug resistance protein 2 (MRP2), mediates this neutrophil influx. Information about a possible counterbalance pathway that could signal the lack of or resolution of an apical inflammatory signal, however, has yet to be described. We now report a system with such hallmarks. Specifically, we identify endocannabinoids as the first known endogenous substrates of the apically restricted multidrug resistance transporter P-glycoprotein (P-gp) and reveal a mechanism, which we believe is novel, for endocannabinoid secretion into the intestinal lumen. Knockdown or inhibition of P-gp reduced luminal secretion levels of N-acyl ethanolamine-type endocannabinoids, which correlated with increased neutrophil transmigration in vitro and in vivo. Additionally, loss of CB2, the peripheral cannabinoid receptor, led to increased pathology and neutrophil influx in models of acute intestinal inflammation. These results define a key role for epithelial cells in balancing the constitutive secretion of antiinflammatory lipids with the stimulated secretion of proinflammatory lipids via surface efflux pumps in order to control neutrophil infiltration into the intestinal lumen and maintain homeostasis in the healthy intestine.

Development of angioimmunoblastic T-cell lymphoma after treatment of diffuse large B-cell lymphoma: a case report and review of literature.

Cases of diffuse large B-cell lymphoma (DLBCL) arising after the initial diagnosis of angioimmunoblastic T-cell lymphoma (AITL) and DLBCL synchronous with AITL have been reported. To date, there is no report on the subsequent development of AITL in patients with DLBCL. Here we presented a rare case of AITL developing six months after the initial diagnosis of DLBCL. In order to investigate the clinical and molecular features of patients with AITL and DLBCL, we also reviewed the literature on AITL patients developing DLBCL, and patients with composite AITL and DLBCL.

Amelioration of atherosclerosis by tanshinone IIA in hyperlipidemic rabbits through attenuation of oxidative stress.

Oxidative stress plays a crucial role in atherogenesis, which raises the possibility of using antioxidants to ameliorate atherosclerosis. In the present study, we aim to determine the effects of tanshinone IIA (TSIIA) on atherosclerosis in hyperlipidemic rabbits. After feeding the rabbits on a high-lipid diet for 90 days, they developed severe atherosclerotic lesions both morphologically and biochemically and exhibited significantly elevated serum lipid, malondialdehyde (MDA) and oxidized low density lipoprotein (oxLDL) levels. Oral administration of TSIIA (3-30 mg/kg) greatly inhibited the formation of atherosclerotic lesions. In TSIIA-treated rabbits, there was a marked reduction in serum and aortic lipid peroxide product content, represented by MDA and oxLDL, whereas enhanced activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) were observed. However, TSIIA had no effect on serum lipid profiles. These results suggest that TSIIA attenuates oxidative stress by decreasing oxLDL production and enhancing activities of SOD and GPx, which might be contributed to the amelioration of atherosclerosis.

[Construction and expression analysis of micro-linear vector as a new general gene therapy vector].

The most difficult field in gene therapy is that vector system should offer both a means of successful transfection and a maximum of safety for the patient. Viral vectors and plasmid vectors are traditional vectors; they may cause unwanted immunological side effects resulting from the expression of nontherapeutic genes. Our aim is to develop a new general gene therapy vector which is suggested to be called as Micro-Linear Vector. The gene expression cassette is capped by our designed cap, including promoter, enhancer, objective gene, and RNA-stabilizing sequence, so it can defend the exnuclease in the eukaryotic cell, at the same time, DNA not encoding the objective gene is reduced to a minimum. The GFP gene is separated from the pEGFP-N3 plasmid, and acts as a reporter gene to construct the Micro-Linear Vector, then both the new vector and the plasmid are transfected to cells, the results are tested by fluorescence microscope and flow cytometry. The results show that the Micro-Linear Vector has a high effective of transfection and safety in 293, 3T3, CNE2 and B95-8 cell lines, at the same time it is less toxicity than the plasmid. We can get the rudiments of conclusion that Micro-Linear Vector has high effection of the transfection and more safety than tradition plasmid in eukaryotic cell.

[Preparation of anti-human indoleamine 2,3-dioxygenase polyclonal antibody].

BACKGROUND & OBJECTIVE: Indoleamine 2,3-dioxygenase (IDO), a cytosolic hemoprotein, catalyzes the rate-limiting step in tryptophan catabolism along the kynurenine pathway in mammals, arrests the growth of pathogens, and suppresses T-cell responses, therefore, leads to IDO-dependent tumor immune tolerance. This study was to express and purify His-hIDO fusion protein and to generate rabbit anti-human IDO polyclonal antibody, which was used to analyze IDO expression in tumor cells. METHODS: Human IDO cDNA was cloned into pET30a(+). The recombinant vector pET30a(+)-hIDO was transformed into BL21 after sequencing. The expression of His-hIDO protein was induced by IPTG. The anti-human IDO polyclonal antibody was obtained by immunizing rabbits with purified His-hIDO protein. The quality of the antibody was identified by Western blot. IDO expression in human fibroblast cancer cell line A431 and liver cancer cell line HepG2 induced by interferon-gamma (IFN-gamma) was also analyzed using the antibody. RESULTS: His-hIDO fusion protein was specifically combined with His-probe polyclonal antibody. The rabbit anti-human IDO polyclonal antibody was of high titer with high specificity. It could recognize IDO expression induced by IFN-gamma in A431 and HepG2 cells. CONCLUSION: The rabbit anti-human IDO polyclonal antibody could recognize IDO expression in tumor cells in vitro effectively, therefore, provides a tool to study the role of IDO in tumor immune tolerance.