No abstract

Pancreatic ductal adenocarcinoma (PDA) is characterized by its highly immunosuppressive tumor microenvironment (TME) that limits T cell infiltration and induces T cell hypofunction. Mesothelin-redirected chimeric antigen receptor T cell (meso-CAR T cell) therapy has shown some efficacy in clinical trials but antitumor efficacy remains modest. We hypothesized that combined meso-CAR T cells with an oncolytic adenovirus expressing TNF-α and IL-2 (Ad5/3-E2F-D24-TNFa-IRES-IL2, or OAd-TNFa-IL2) would improve efficacy. OAd-TNFa-IL2 enhanced the antitumor efficacy of meso-CAR T cells in human-PDA-xenograft immunodeficient mice and efficacy was associated with robustly increased tumor-infiltrating lymphocytes (TILs), enhanced and prolonged T cell function. Mice treated with parental OAd combined with meso-CAR T developed tumor metastasis to the lungs even if primary tumors were controlled. However, no mice treated with combined OAd-TNFa-IL2 and meso-CAR T died of tumor metastasis. We also evaluated this approach in a syngeneic mouse tumor model by combining adenovirus expressing murine TNF-α and IL-2 (Ad-mTNFa-mIL2) and mouse CAR T cells. This approach induced significant tumor regression in mice engrafted with highly aggressive and immunosuppressive PDA tumors. Ad-mTNFa-mIL2 increased both CAR T cell and host T cell infiltration to the tumor and altered host tumor immune status with M1 polarization of macrophages and increased dendritic cell maturation. These findings indicate that combining cytokine-armed oncolytic adenovirus to enhance the efficacy of CAR T cell therapy is a promising approach to overcome the immunosuppressive TME for the treatment of PDA.

T cells expressing chimeric antigen receptors (CART) have shown significant promise in clinical trials to treat hematologic malignancies, but their efficacy in solid tumors has been limited. Oncolytic viruses have the potential to act in synergy with immunotherapies due to their immunogenic oncolytic properties and the opportunity of incorporating therapeutic transgenes in their genomes. Here, we hypothesized that an oncolytic adenovirus armed with an EGFR-targeting, bispecific T-cell engager (OAdBiTE) would improve the outcome of CART-cell therapy in solid tumors. We report that CART cells targeting the folate receptor alpha (FR-a) successfully infiltrated preestablished xenograft tumors but failed to induce complete responses, presumably due to the presence of antigen-negative cancer cells. We demonstrated that OAd-BiTE-mediated oncolysis significantly improved CART-cell activation and proliferation, while increasing cytokine production and cytotoxicity, and showed an in vitro favorable safety profile compared with EGFR-targeting CARTs. BiTEs secreted from infected cells redirected CART cells toward EGFR in the absence of FR-a, thereby addressing tumor heterogeneity. BiTE secretion also redirected CAR-negative, nonspecific T cells found in CART-cell preparations toward tumor cells. The combinatorial approach improved antitumor efficacy and prolonged survival in mouse models of cancer when compared with the monotherapies, and this was the result of an increased BiTEmediated T-cell activation in tumors. Overall, these results demonstrated that the combination of a BiTE-expressing oncolytic virus with adoptive CART-cell therapy overcomes key limitations of CART cells and BiTEs as monotherapies in solid tumors and encourage its further evaluation in human trials.

AQP4 deletion in mice is neuroprotective in a transient ischemia model of retinal injury, suggesting the possible use of AQP4 inhibitors in retinal vascular occlusive and ischemic diseases.

Objective Mitochondrial biology appears central to many conditions that progress to death but remains poorly characterized following cardiac arrest. Mitochondrial dysfunction in electron transfer and reactive oxidant species (ROS) leakage during ischemia may lead to downstream events including mitochondrial protein oxidation, tyrosine nitrosylation, cytochrome c loss, and eventual death. We sought to better define early fixed alterations in these mitochondrial functions following whole animal cardiac arrest. Methods We used a murine model of 8 minutes of untreated KCl-induced cardiac arrest followed by resuscitation and return of spontaneous circulation (ROSC) to study mitochondrial functions in four groups of animals: (a) after 8 min cardiac arrest (CA8) but no resuscitation, (b) 30 min post-ROSC (R30), (c) 60 min post-ROSC (R60) and in (d) shams. Heart mitochondria were immediately harvested, isolated and stored at −80°C for later spectrophotometric measurements of electron transfer activities and ROS leakage using appropriate substrates and inhibitors. Mitochondrial cytochrome c content and tyrosine nitration were analyzed by western blot and densitometry. Results A significant ROS leakage from Complex I was evident after just 8 min of cardiac arrest (CA8 group, P<0.05), which was followed by a progressive reduction in Complex I electron transfer activity (CA8>R30>R60). In contrast, Complex II and II–III activities appeared more resistant to ischemia at the time points evaluated. Early changes in a ~50 kDa and ~25 kDa protein were observed in tyrosine nitration along with a loss of cytochrome c. Conclusions A relatively “orderly” process of mitochondrial dysfunction progresses during ischemia and reperfusion. Changes in mitochondrial ROS generation and electron transfer from Complex I occur along with tyrosine nitrosylation and loss of cytochrome c; these may represent important new targets for future human therapies.