ALBERT

Description: In this article, we report an electroless method to fabricate porous hexagonal silicon carbide and hexagonal silicon carbide nanoparticles (NPs) as small as 1 nm using wet chemical stain etching. We observe quantum confinement effect for ultrasmall hexagonal SiC NPs in contrast to the cubic SiC NPs. We attribute this difference to the various surface terminations of the two polytypes of SiC NPs.

Description: Small food retailers, including corner/convenience stores, pharmacies, gas-marts, and dollar stores, have historically stocked limited fruits and vegetables, though this may be changing. We examined increases in sales, customer purchasing, and stocking of fresh and/or frozen fruits and vegetables in small food stores over time and in relation to: (a) a local food policy (the Minneapolis Staple Foods Ordinance) and (b) neighborhood socioeconomic status (SES). We used longitudinal data (2014–2017) from 147 randomly-sampled stores in Minneapolis/St. Paul, USA, collected using interviewer-administered manager surveys (measuring sales and stocking) and customer intercepts/observations (measuring purchasing, n = 3039). The local policy required Minneapolis stores to meet minimum stocking standards for fresh/frozen produce and other healthy foods. No ordinance existed in St. Paul. Mixed regression models examined overall change over time and change by city and neighborhood SES. We observed significant increases over time (p 〈 0.05) in sales and purchasing of fresh fruit and in stocking of fresh fruit, frozen fruit, and frozen vegetables. We did not identify consistent statistical evidence for differential change in sales, purchasing, or stocking by city or neighborhood SES. Key study findings suggest limited differential effects of the local ordinance and/or neighborhood SES. However, findings also indicate significant time trends for some products, including consistent improvements in sales, customer purchasing, and stocking of fresh fruit. Given the ready-to-eat convenience of many fresh fruits and their broad appeal, fresh fruit appears a promising target for advancing the healthfulness of small food retailers.

Description: Abstract 2188 25%-30% of patients with hemophilia A develop neutralizing antibodies following replacement therapy with factor VIII (FVIII). These patients can be treated with factor VIIa (FVIIa) which triggers the extrinsic pathway of coagulation and thereby bypasses the requirement for FVIII. We developed a new mouse model that is transgenic for human FVII and expresses specific immune tolerance to native human FVIIa. We aim to investigate the immunological impact of modified FVIIa product candidates and to characterize their immunogenicity by analyzing emerging FVIIa-specific T cell responses. The new mouse model offers a unique opportunity to study central and peripheral immune regulatory mechanisms and the generation of immune responses by pro-inflammatory antigen-specific effector T cells (Teff). We hypothesized that FVIIa-specific Teff having escaped clonal deletion are present in the periphery and may be actively suppressed by FVIIa-specific regulatory T cells (Tregs). To study this hypothesis, we immunized mice with recombinant FVIIa (rFVIIa) with or without LPS, a well-described “danger signal” being able to break immune tolerance by stimulating the innate immune system. Intravenous or subcutaneous administration of rFVIIa alone did not elicit antibody responses and thus immune tolerance to rFVIIa was not broken. However, co-administration of rFVIIa and LPS resulted in a specific antibody response that was not isotypically restricted. To further analyze the mechanisms behind this break of specific immune tolerance, we characterized rFVIIa-specific T cells by the expression of CD154, a marker of antigen-specific T cells. Cytokine production and CD154 expression were assessed upon re-stimulation with rFVIIa. In contrast to mice that were immunized with rFVIIa only, we found increased numbers of rFVIIa-specific T cells in rFVIIa-LPS-treated mice displaying a stable, highly pro-inflammatory (IL-2+/IFN-g+) memory phenotype. These data could suggest that rFVIIa-specific Teff that escaped clonal deletion during induction of central immune tolerance, are present in the periphery of human FVII-transgenic mice. This would imply that rFVIIa-specific Teff could be actively suppressed by Tregs. This suppression could be overcome by danger signals like LPS. We currently study the regulatory mechanisms that maintain tolerance upon administration of FVIIa without LPS. We are approaching this question by correlating the characteristics of FVIIa-specific Teff and Treg responses under both tolerant and non-tolerant conditions. Ultimately, we aim to understand which danger signals have to be provided to break immune tolerance and how tolerance is regulated. Understanding these regulatory mechanisms will enable us to develop new therapeutic strategies and prevent conditions that lead to the induction of antibodies against drug products in patients. Disclosures: Lenk: Baxter BioScience: Employment. Pasztorek:Baxter BioScience: Employment. Weiller:Baxter BioScience: Employment. Ahmad:Baxter BioScience: Employment. Schwarz:Baxter BioScience: Employment. Scheiflinger:Baxter BioScience: Employment. Reipert:Baxter Innovations GmbH: Employment. de la Rosa:Baxter BioScience: Employment.

Description: Despite the advances in the treatment of pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL), infiltration of the central nervous system (CNS) remains a clinical challenge. Certain cytogenetic subtypes such as E2A-PBX1-and BCR-ABL-positive BCP-ALL confer a higher risk for CNS involvement initially and for CNS relapse. Novel strategies to predict CNS and to eradicate leukemic cells from the CNS are subjects of ongoing research. In order to identify targets with diagnostic and therapeutic relevance, comparative RNA-sequencing was performed with patient derived xenograft (PDX) blasts from 5 E2A-PBX1-positive patients, recovered from the bone marrow (BM) and from the CNS of NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Differential gene expression analysis revealed the upregulation of various genes of the pre-B cell receptor complex, particularly the signaling component CD79a (Igα) in blasts recovered from the CNS as compared to blasts from the BM. We then investigated the impact of CD79a on CNS infiltration in vivo and in patients. CD79a was downregulated by short-hairpin RNA (shRNA) mediated knockdown in the E2A-PBX1 positive cell line 697. Proliferation rates of 697-shCD79a cells and control-transfected 697 cells (697-shCtrl) in vitro were similar. Furthermore, NSG mice injected with 697-shCD79a cells showed comparable survival times, as well as similar blast infiltration in spleen and BM as animals injected with 697-shCtrl cells. However, downregulation of CD79a led to a significantly lower number of CNS-positive mice (4/15, 26%) as compared to control animals (7/10, 70%) (p=0.0486, Figure A). This indicates that CD79a is not critically involved in proliferation and peripheral engraftment, but in CNS infiltration of E2A-PBX1 positive 697 cells in vivo. To test if CD79a also affects CNS involvement in BCR-ABL-positive leukemia, a murine/murine transplantation model was used. B-cells isolated from CD79a-knockout (CD79a-KO) or wildtype mice (CD79a-Ctrl) were stably transfected with a BCR-ABL fusion gene and cultured independent of cytokines, thereby inducing malignant transformation. Both cell lines were subsequently injected into recipient NSG mice (n=8/group) and leukemic development was followed. The experiment was terminated when all control mice had developed leukemic symptoms and mice were analyzed for leukemic engraftment. A further CD79a-KO group was included for survival analysis. Median spleen volume as a surrogate of leukemic infiltration was significantly lower in mice injected with CD79a-KO as compared to CD79a-Ctrl cells (0.35 cm³ vs. 0.06 cm³; p=0.0001). Median blast percentages in spleens and BM were also markedly reduced (75.3% vs. 5.8%; p=0.0001 and 61.0% vs. 4.5%; p=0.0001, respectively). Importantly, none of the animals in the CD79a-KO group showed blasts in the CNS as assessed by histology whereas blasts were present in all of the animals in the CD79a-Ctrl group. Finally and most importantly, NSG-mice injected with CD79a-KO cells showed a highly significant prolongation in median survival as compared to mice with CD79a-Ctrl cells (29 days vs. 95 days; p=0.0001, Figure B). Altogether, these data suggest that in a model of BCR-ABL-positive leukemia, absence of CD79a impacts the engraftment of blasts in vivo, in the CNS and other leukemic niches. To further validate our findings in patient material, we measured CD79a protein expression in PDX cells from an E2A-PBX1- and a BCR-ABL-positive patient serially transplanted into NSG mice for three passages. For both entities and in all passages, CNS blasts showed a higher CD79a expression than blasts isolated from the bone marrow. In order to assess if CD79a can be used as a marker to predict CNS involvement in patients, CD79a mRNA levels were measured in a selected cohort of 98 pediatric BCP-ALL patients, which contained 26 CNS-positive patients matched to 72 CNS-negative patients. CNS-positive patients showed significantly higher mRNA levels of CD79a than CNS-negative patients (p=0.0225, unpaired t-test, Figure C) suggesting that CD79a may be of value as a potential diagnostic marker for initial CNS involvement in BCP-ALL. Our results indicate a role of CD79a in proliferation and CNS infiltration of BCP-ALL blasts in experimental settings and patients. We intend to prospectively evaluate CD79a as a prognostic marker, which may also be a therapeutic target in CNS-positive BCP-ALL. Disclosures No relevant conflicts of interest to declare.

Description: The application of antibodies is a promising option in the treatment of B-cell malignancies, including acute lymphoblastic leukemia (ALL) and B-cell Non-Hodgkin lymphoma (B-NHL). Although patient outcomes have improved by applying combinations of chemotherapy and antibodies, certain patients characterized by a high expression of anti-apoptotic Bcl-2 have a poor prognosis. These include adult B-NHL patients with "double-hit lymphomas" (DHLs) and pediatric ALL patients harboring a t(17;19) translocation. Furthermore, a substantial number of Burkitt´s lymphoma (BL) patients also express Bcl-2 even though the impact of this finding on prognosis is yet unclear. Here, we examine the role of low doses of the Bcl-2 inhibitor venetoclax (VTX, 1nM) on the efficacy of the therapeutic antibodies rituximab (CD20), daratumumab (CD38) and CD19-DE (a variant of the CD19 antibody MOR208 engineered for improved effector cell binding). Natural killer (NK)-cell mediated antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) were evaluated. The CD20-expressing DHL cell line Carnaval and patient-derived xenograft (PDX) cells from two BL patients were used as target cells for rituximab, the CD20-negative/CD38-positive DHL cell line Will-2 was used with daratumumab and three PDX samples from t(17;19) positive ALL patients were used with CD19-DE. For the assessment of ADCP, human monocyte-derived macrophages were incubated with labelled target cells and microscopy assays were performed. NK-cell mediated ADCC was not enhanced by VTX in any of our models. However, 17-37% increases in ADCP by macrophages were detected when Carnaval cells were subjected to combinations of VTX/rituximab and when Will-2 cells were treated with VTX/daratumumab as compared to VTX or antibody alone (p=0.0318/p=0.0185 and p=0.0012/p=0.0068, respectively, Figure A). When BL PDX cells were subjected to ADCP assays with VTX/rituximab, mean phagocytosis levels were also enhanced by 26.0% and 21.0% in the combination treatment group as compared to VTX (p=0.0283) and rituximab alone (p=0.0282; Figure A). ADCP assays with t(17;19) positive ALL-PDX cells and CD19-DE confirmed these results as phagocytosis was increased to similar extents in the combination group as compared to VTX (p=0.0017) or CD19-DE alone (p=0.0323) (Figure A/B). In order to exclude that our observations were due to an enhancement of apoptosis in target cells only, we measured cleaved caspase-3 with VTX, antibodies alone or the combination of both. Cleaved caspase-3 levels were equal in all groups suggesting that the addition of VTX resulted in an apoptosis-independent activation of macrophages. In order to minimize heterogeneity in ADCP assays, phagocytosis was next examined using expanded macrophages from NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice for our assays. Regardless of target cells and antibody, the combination of antibody treatment with VTX resulted in enhanced phagocytosis by murine macrophages, confirming our results. The effects of VTX on the efficacy of rituximab were finally examined in vivo. Carnaval cells were injected intravenously into NSG mice and animals treated with VTX (100 mg/kg 5 days/week by oral gavage), rituximab alone (1 mg/kg once weekly intraperitoneally) or the combination of both (n=6/group). Mice were sacrificed when mice showed clinical lymphoma or leukemia engraftment and survival differences were assessed using Kaplan-Meier log-rank statistics. Compared to control, mice treated with VTX displayed a slight survival advantage, which was more marked in mice treated with rituximab (p=0.0020/p=0.0004, respectively, Figure C), suggesting a better efficacy of rituximab than VTX as monotherapy. Most importantly, mice treated with the combination VTX/rituximab showed significantly superior survival as compared to either VTX or rituximab alone (p=0.0023/p=0.0268, respectively, Figure C), suggesting additive effects in vivo. Altogether, we show that VTX enhances the efficacy of therapeutic antibodies in models of B-cell malignancies including PDX samples. The mechanism is most likely dependent on distinct influences of VTX on macrophage activation, e. g. by myeloid immune checkpoints. Our in vivo data suggest that this combination strategy may become a promising therapeutic option for the treatment of Bcl-2 expressing B-cell malignancies in the future. Figure. Figure. Disclosures Bourquin: Amgen: Other: Travel Support. Valerius:Affimed: Research Funding. Peipp:Affimed: Research Funding.

Description: Brachypodium distachyon is an established model for drought tolerance. We previously identified accessions exhibiting high tolerance, susceptibility and intermediate tolerance to drought; respectively, ABR8, KOZ1 and ABR4. Transcriptomics and metabolomic approaches were used to define tolerance mechanisms. Transcriptional analyses suggested relatively few drought responsive genes in ABR8 compared to KOZ1. Linking these to gene ontology (GO) terms indicated enrichment for “regulated stress response”, “plant cell wall” and “oxidative stress” associated genes. Further, tolerance correlated with pre-existing differences in cell wall-associated gene expression including glycoside hydrolases, pectin methylesterases, expansins and a pectin acetylesterase. Metabolomic assessments of the same samples also indicated few significant changes in ABR8 with drought. Instead, pre-existing differences in the cell wall-associated metabolites correlated with drought tolerance. Although other features, e.g., jasmonate signaling were suggested in our study, cell wall-focused events appeared to be predominant. Our data suggests two different modes through which the cell wall could confer drought tolerance: (i) An active response mode linked to stress induced changes in cell wall features, and (ii) an intrinsic mode where innate differences in cell wall composition and architecture are important. Both modes seem to contribute to ABR8 drought tolerance. Identification of the exact mechanisms through which the cell wall confers drought tolerance will be important in order to inform development of drought tolerant crops.

Description: Comprehensive large-scale pharmacovigilance surveillances are very effective tools to collect data on products in the post authorization period. The evaluation presented here was set up to assess the long-term efficacy, safety and tolerability of the recombinant FVIII concentrate, Helixate®NexGen (in the US: Helixate®FS). Sixty-five hemophilia centers in Germany, Austria, Italy, France, and Sweden are participating in this ongoing international project. Previously untreated (PUP) and previously treated patients (PTP) at any age suffering from hemophilia A treated with Helixate®NexGen were eligible for the surveillance. Based on the standard schedule preferred at the centers, patients are routinely screened every 3 to 12 months. At these visits, the following parameters, as used routinely for these patients, were documented (non-interventional design): overall clinical response, pharmacokinetics, occurrence of bleedings, adverse drug reactions including the incidence of inhibitors, other laboratory safety parameters, coagulation parameters, relevant concomitant diseases, and medication. To determine the level of exposure, treatment modalities with Helixate®NexGen, including average factor consumption per month and exposure days are recorded. One hundred and ten patients have been included into the investigation up to now; data from 104 patients were available for this interim analysis including 2 PUPs. The median age was 25 years (range: 8 months – 68 years). One patient suffered from mild, eleven from moderate, and 89 from severe hemophilia A; in three patients the information on severity was missing. Most of the patients were treated prophylactically (92%, at least one infusion per week). Exposure to Helixate®NexGen during the pharmacovigilance ranged between 5 and 703 days. A total of 112 bleedings were documented during the observation period, with a range of 0 to 20 bleedings per patient per year. Efficacy of Helixate®NexGen was assessed as good or excellent in 97% of all cases. There were no cases of inhibitor development during the 2-year observation period, especially no cases of inhibitors when patients were switched from products produced in Chinese hamster ovary (CHO) cells to Helixate®NexGen, which is produced in baby hamster kidney (BHK) cells. The good tolerability of Helixate®NexGen was further supported by the fact that there were no other relevant side effects documented. In summary this pharmacovigilance supports the good tolerability and good/excellent efficacy data of Helixate®NexGen.

Description: Abstract 3327 Baxter has developed a recombinant FVIIa (rFVIIa) product for the treatment of hemophilia patients with factor VIII or factor IX inhibitors. Before entering clinical development we assessed the immunogenic safety profile of the new product candidate BAX 817 using two novel mouse models that mimicked specific aspects of the situation in patients. Several comparative preclinical immunogenicity studies were conducted to assess the immunogenicity profile of Baxter`s rFVIIa BAX 817 in comparison to a licensed recombinant FVIIa product. Three different mouse models were used for this purpose. The first model expresses specific immune tolerance against human FVIIa and, therefore, mimics the situation in both hemophilia A and hemophilia B patients. Using this model, we asked if Baxter‘s rFVIIa is able to maintain immune tolerance to human FVIIa. The second model is a hemophilia A model that mimics the situation in an important fraction of hemophilia A patients with FVIII inhibitors. This model expresses a human MHC-class II haplotype (HLA-DRB1*1501) that was previously shown to be associated with an increased risk for the development of FVIII inhibitors. The third model represents a normal wildtype C57BL/6 mouse. All mice were treated with 8 weekly doses of either Baxter‘s rFVIIa BAX 817 or a licensed recombinant FVIIa product. Total anti-FVIIa antibodies were analyzed prior to the first dose as well as after the 4th and the 8th dose using ELISA assays. Baxter`s rFVIIa BAX 817 and a licensed recombinant FVIIa product induced similar titers of anti-FVIIa antibodies in C57BL/6 wildtype mice and in hemophilic HLA-DRB1*1501 mice. In addition, both Baxter`s rFVIIa BAX 817 and a licensed recombinant FVIIa product were able to maintain specific immune tolerance in a novel mouse model that is immunologically tolerant to human FVIIa. Based on the data obtained we conclude that both Baxter‘s rFVIIa BAX 817 and a licensed recombinant FVIIa product have a comparable immunogenic safety profile. Disclosures: Horling: Baxter Innovations GmbH: Employment. Lenk:Baxter BioScience: Employment. Ahmad:Baxter BioScience: Employment. Weiller:Baxter BioScience: Employment. Schuster:Baxter Innovation GmbH: Employment. Schwarz:Baxter Innovations GmbH: Employment. Reipert:Baxter Innovations GmbH: Employment.