There is a Blood Commentary on this article in this issue.

ß-thalassemia intermedia is a disorder characterized by ineffective erythropoiesis (IE), anemia, splenomegaly and systemic iron overload. Novel approaches are being explored based on the modulation of pathways that reduce iron absorption (i.e. using hepcidin activators like Tmprss6-antisense oligonucleotides (ASO)) or increase erythropoiesis (by erythropoietin (EPO) administration or by modulating the ability of transferrin receptor 2 (Tfr2) to control red blood cell (RBC) synthesis). Targeting Tmprss6 mRNA by Tmprss6-ASO was proven to be effective in improving the IE and splenomegaly by inducing iron restriction. However we postulated that combinatorial strategies might be superior to single therapies. Here we combined Tmprss6-ASO with EPO administration or removal of a single Tfr2 allele in the bone marrow of animals affected by ß-thalassemia intermedia (Hbbth3/+). EPO administration alone or removal of a single Tfr2 allele increased hemoglobin levels and RBCs. However, EPO or Tfr2 single allele deletion alone, respectively, exacerbated or did not improve the splenomegaly in ß-thalassemic mice. To overcome this issue, we postulated that some level of iron restriction (by targeting Tmprss6) would improve the splenomegaly while preserving the beneficial effects on RBC production mediated by EPO or Tfr2 deletion. While administration of Tmprss6-ASO alone improved the anemia, combination of Tmprss6-ASO+EPO or Tmprss6-ASO+Tfr2 single allele deletion showed significantly higher hemoglobin levels as well as reduction of splenomegaly. In conclusion, our results clearly indicate that these combinatorial approaches are superior to single treatments in ameliorating the IE and anemia in ß-thalassemia and could provide guidance to translate some of these approaches into viable therapies.

Short-term administration of JAK2 inhibitors reduces splenomegaly in mouse models of b-thalassemia intermedia and major b-thalassemia is one of the most common congenital red blood cell (RBC) disorders characterized by limited bglobin synthesis and ineffective erythropoiesis.

Hematopoietic cell transplantation (HCT) is a last resort, potentially curative treatment option for pediatric patients with refractory acute myeloid leukemia (AML). Cord blood transplantation (CBT) results in less relapses and less graft-versus-host disease when compared to other sources. Nevertheless, still more than half of the children die from relapses. We therefore designed a strategy to prevent relapses by inducing anti-AML immunity after CBT, using a CB-derived dendritic cell (CBDC) vaccine generated from CD34+ CB cells from the same graft. We here describe the optimization and validation of good manufacturing practice (GMP)-grade production of the CBDC vaccine. We show the feasibility of expanding low amounts of CD34+ cells in a closed bag system to sufficient DCs per patient for at least three rounds of vaccinations. The CBDCs showed upregulated costimulatory molecules after maturation and showed enhanced CCR7-dependent migration toward CCL19 in a trans-well migrations assay. CBDCs expressed Wilms' tumor 1 (WT1) protein after electroporation with WT1-mRNA, but were not as potent as CBDCs loaded with synthetic long peptides (peptivator). The WT1-peptivator loaded CBDCs were able to stimulate T-cells both in a mixed lymphocyte reaction as well as in an antigen-specific (autologous) setting. The autologous stimulated T-cells lysed not only the WT1+ cell line, but most importantly, also primary pediatric AML cells. Altogether, we provide a GMP-protocol of a highly mature CBDC vaccine, loaded with WT1 peptivator and able to stimulate autologous T-cells in an antigen-specific manner. Finally, these Tcells lysed primary pediatric AML demonstrating the competence of the CBDC vaccine strategy.

BackgroundImmunotherapy in high-risk neuroblastoma (HR-NBL) does not live up to its full potential due to inadequate (adaptive) immune engagement caused by the extensive immunomodulatory capacity of HR-NBL. We aimed to tackle one of the most notable immunomodulatory processes in neuroblastoma (NBL), absence of major histocompatibility complex class I (MHC-I) surface expression, a process greatly limiting cytotoxic T cell engagement. We and others have previously shown that MHC-I expression can be induced by cytokine-driven immune modulation. Here, we aimed to identify tolerable pharmacological repurposing strategies to upregulate MHC-I expression and therewith enhance T cell immunogenicity in NBL.MethodsDrug repurposing libraries were screened to identify compounds enhancing MHC-I surface expression in NBL cells using high-throughput flow cytometry analyses optimized for adherent cells. The effect of positive hits was confirmed in a panel of NBL cell lines and patient-derived organoids. Compound-treated NBL cell lines and organoids were cocultured with preferentially expressed antigen of melanoma (PRAME)-reactive tumor-specific T cells and healthy-donor natural killer (NK) cells to determine the in vitro effect on T cell and NK cell cytotoxicity. Additional immunomodulatory effects of histone deacetylase inhibitors (HDACi) were identified by transcriptome and translatome analysis of treated organoids.ResultsDrug library screening revealed MHC-I upregulation by inhibitor of apoptosis inhibitor (IAPi)- and HDACi drug classes. The effect of IAPi was limited due to repression of nuclear factor kappa B (NFκB) pathway activity in NBL, while the MHC-I-modulating effect of HDACi was widely translatable to a panel of NBL cell lines and patient-derived organoids. Pretreatment of NBL cells with the HDACi entinostat enhanced the cytotoxic capacity of tumor-specific T cells against NBL in vitro, which coincided with increased expression of additional players regulating T cell cytotoxicity (eg, TAP1/2 and immunoproteasome subunits). Moreover, MICA and MICB, important in NK cell cytotoxicity, were also increased by entinostat exposure. Intriguingly, this increase in immunogenicity was accompanied by a shift toward a more mesenchymal NBL cell lineage.ConclusionsThis study indicates the potential of combining (immuno)therapy with HDACi to enhance both T cell-driven and NKcell-driven immune responses in patients with HR-NBL.

Recent developments in gene engineering technologies have drastically improved the therapeutic treatment options for cancer patients. The use of effective chimeric antigen receptor T (CAR-T) cells and recombinant T cell receptor engineered T (rTCR-T) cells has entered the clinic for treatment of hematological malignancies with promising results. However, further fine-tuning, to improve functionality and safety, is necessary to apply these strategies for the treatment of solid tumors. The immunosuppressive microenvironment, the surrounding stroma, and the tumor heterogeneity often results in poor T cell reactivity, functionality, and a diminished infiltration rates, hampering the efficacy of the treatment. The focus of this review is on recent advances in rTCR-T cell therapy, to improve both functionality and safety, for potential treatment of solid tumors and provides an overview of ongoing clinical trials. Besides selection of the appropriate tumor associated antigen, efficient delivery of an optimized recombinant TCR transgene into the T cells, in combination with gene editing techniques eliminating the endogenous TCR expression and disrupting specific inhibitory pathways could improve adoptively transferred T cells. Armoring the rTCR-T cells with specific cytokines and/or chemokines and their receptors, or targeting the tumor stroma, can increase the infiltration rate of the immune cells within the solid tumors. On the other hand, clinical “off-tumor/on-target” toxicities are still a major potential risk and can lead to severe adverse events. Incorporation of safety switches in rTCR-T cells can guarantee additional safety. Recent clinical trials provide encouraging data and emphasize the relevance of gene therapy and gene editing tools for potential treatment of solid tumors.

Antisense technology is a powerful drug discovery approach for identifying oligonucleotide analogs that can specifically modify RNA expression through multiple mechanisms including RNase H1-mediated degradation of RNA and modulation of RNA splicing. We have successfully applied this technology towards targeting a number of transcripts in a wide-range of therapeutic areas. Beta-thalassemia, one of the most common genetic disorders worldwide, is characterized by reductions in beta-globin and ineffective erythropoiesis. This in turn leads to suppression of hepcidin, a peptide hormone that serves as the master regulator of iron homeostasis. Inappropriately low levels of hepcidin trigger increased dietary iron absorption resulting in iron overload, which is the major cause of morbidity and mortality in beta-thalassemia patients. TMPRSS6 is a transmembrane serine protease mainly produced by hepatocytes that negatively regulates hepcidin expression. Previous mouse and human genetic data from multiple groups suggest that lowering TMPRSS6 expression could up-regulate hepcidin and ameliorate many of the disease symptoms associated with beta-thalassemia. We identified potent antisense oligonucleotides (ASOs) against mouse TMPRSS6. Downregulation of TMPRSS6 with ASO treatment results in dose-dependent hepcidin upregulation, which leads to dramatic reductions in serum iron and transferrin saturation. This in turn ameliorated the anemia and iron overload phenotypes in a mouse model of beta-thalassemia (th3/+ mice), which recapitulates beta-thalassemia intermedia in humans (Guo et al. J Clin Invest. 2013; 123(4):1531-41). Moreover, this ASO can be combined efficiently with iron chelators for the management of iron overload and anemia in non-transfusion-dependent thalassemia (Casu et al. Haematologica. 2016; 101(1):e8-e11). TMPRSS6 is predominantly expressed in hepatocytes, for which we have developed a targeted delivery approach with triantennary N-acetyl galactosamine (GalNAc). With GalNAc-conjugated ASOs, a ~10-fold improvement in potency is observed for many liver targets (Prakash et al. Nucleic Acids Res. 2014; 42(13):8796-807). In order to characterize GalNAc-conjugated TMPRSS6 ASO, we treated normal mice with both parent ASO and its conjugated counterpart. As expected, the conjugated ASO demonstrated a ~10-fold improvement in ED50 (25 mg/kg/week versus 2.5 mg/kg/week for parent and conjugated ASOs, respectively). Next, we treated th3/+ mice for six weeks with 10 mg/kg/week GalNAc-conjugated TMPRSS6 ASO or a control ASO of the same chemistry. Compared to the control ASO treatment group, we observed >95% reduction of TMPRSS6 mRNA levels and >3-fold up-regulation of hepcidin mRNA levels in the liver. This resulted in a ~40% reduction in serum iron and ~50% reduction in transferrin saturation. In addition, anemia phenotypes were significantly improved as shown by a significant increase in hemoglobin and red blood cells (from 7.0 g/dL to 8.9 g/dL and from 5.8 to 7.5x10^6 cells/µl in the control ASO treatment group and in the TMPRSS6 ASO treatment group, respectively). Furthermore, there was an approximately 50% reduction in spleen weight. Improved erythroid maturation was indicated by a significant reduction in reticulocyte number and a normalized proportion between the pool of erythroblasts and enucleated erythroid cells. A GalNAc-conjugated human TMPRSS6 clinical candidate was identified. Similar to mouse TMPRSS6, GalNAc-conjugated ASO demonstrated superior potency in human primary hepatocyte culture, in human TMPRSS6 transgenic mice and in cynomolgus monkey. Collectively, our data demonstrate that GalNAc-conjugated TMPRSS6 ASO could be an effective therapeutic for patients with beta-thalassemia and related disorders. A Phase 1 clinical trial is planned to initiate in 2017. Disclosures Aghajan: Ionis Pharmaceuticals: Employment, Equity Ownership. Booten:Ionis Pharmaceuticals: Employment, Equity Ownership. Monia:Ionis Pharmaceuticals: Employment, Equity Ownership. Guo:Ionis Pharmaceuticals: Employment, Equity Ownership.

Adoptive T cell therapy utilizing tumor-specific autologous T cells has shown promising results for cancer treatment. However, the limited numbers of autologous tumor-associated antigen (TAA)-specific T cells and the functional aberrancies, due to disease progression or treatment, remain factors that may significantly limit the success of the therapy. The use of allogeneic T cells, such as umbilical cord blood (CB) derived, overcomes these issues but requires gene modification to induce a robust and specific anti-tumor effect. CB T cells are readily available in CB banks and show low toxicity, high proliferation rates, and increased anti-leukemic effect upon transfer. However, the combination of anti-tumor gene modification and preservation of advantageous immunological traits of CB T cells represent major challenges for the harmonized production of T cell therapy products. In this manuscript, we optimized a protocol for expansion and lentiviral vector (LV) transduction of CB CD8 + T cells, achieving a transduction efficiency up to 83%. Timing of LV treatment, selection of culture media, and the use of different promoters were optimized in the transduction protocol. LentiBOOST was confirmed as a non-toxic transduction enhancer of CB CD8 + T cells, with minor effects on the proliferation capacity and cell viability of the T cells. Positively, the use of LentiBOOST does not affect the functionality of the cells, in the context of tumor cell recognition. Finally, CB CD8 + T cells were more amenable to LV transduction than peripheral blood (PB) CD8+ T cells and maintained a more naive phenotype. In conclusion, we show an efficient method to genetically modify CB CD8 + T cells using LV, which is especially useful for off-the-shelf adoptive cell therapy products for cancer treatment.