Research GmbH Research Articles

Multi-tile zinc-oxide-based radiation-hard fast scintillation counter for relativistic heavy-ion beam diagnostics: prototype design and test

This contribution summarizes the design and performance test of a prototype radiation-hard fast scintillation detector based on the indium-doped zinc oxide ceramic scintillator, ZnO(In). The prototype detector has been developed for use as a beam diagnostics tool for high-energy beam lines of the SIS18 synchrotron at the GSI Helmholtz Center for Heavy Ion Research GmbH. The new detector consists of multiple ZnO(In) scintillating ceramics tiles stacked on the front and back sides of a borosilicate light guide. The performance of the detector was tested in comparison to a standard plastic scintillation detector with 300 MeV/u energy 40Ar, 197Au, 208Pb, and 238U ion beams. The investigated prototype exhibits 100% counting efficiency and radiation hardness of a few orders of magnitude higher than the standard plastic scintillation counter. Therefore, it provides an improved beam diagnostics tool for relativistic heavy-ion beam measurements.

GREEN ENERGY IN SMART BUILDINGS INVESTMENTS

The Institute of Radio and Information Systems (IRIS) – Association based in Vienna, Austria, whose staff has extensive experience in scientific work and organizing international conferences in various countries, publishes its own scientific journals and has a book publishing house. We plan to build our actions within the Smart Finance for Smart Buildings (SFSB) initiative and the methodology used in SEI Forums to bring together a wide range of participants, developing it in direction: most of the energy consumed by buildings is spent on space heating including air required for ventilation. Advocacy and development of programs for financing the expansion of the use of air heat pumps with energy recovery of heated (in summer cooled) ventilated air will reduce energy costs for heating by almost 4 times with a quick payback period. In 5 years after project are provided: primary energy savings triggered by the project; investments in sustainable energy triggered by the project; reduction of the greenhouse gases emissions. To create national roundtables on energy efficiency investments as multilateral discussion forums bringing together government, local and regional authorities, financial sector, ESCOs, homeowners, industry, construction sector and SME sector, we propose to engage professionals from our conference partners: Technische Universitaet Wien; IEEE Technology Center GmbH (Wien); Politische Akademie der ÖVP (Wien); Aspern Smart City Research GMBH & CO KG. Our proposals relate mainly to Austria however, they include a share of events in Czech Republic, Slovakia, Hungary, Slovenia which we can successfully organize with help of the IEEE Technology and Engineering Management Society Central Europe Chapter from our partner IEEE Austria Section as well as exchange with roundtables throughout Europe which will be coordinated by Commission services. The proposed project consist of blocks: Information-statistical, Training, Expert, and Legislative initiatives.

Guidelines for rail quality selection to increase service life based on wear and fatigue behavior of various wheel and rail materials

The Austrian rail network is very diverse: The lines in the alpine areas have very tight radii while the lines in the flat areas (east and north of Austria) are mainly straight high-speed lines. This leads to different levels of wheel and rail wear. Accordingly, the objective of this study was to experimentally model a laboratory-scale cyclic wheel-rail contact to investigate wear behavior and rolling contact fatigue at different radii (RCF).A novel test system was set up at AC2T research GmbH to reproduce unidirectional loading on a real track. Tribotests were performed with rail (grades R260, R350HT & R400HT) and wheel (grades ER7 & ER9) parts from the field. This newly designed principle is an effective method to characterize the wheel-rail contact. Wear characterization was done after defined testing cycles to detect (i) area loss and (ii) change in surface topography.

Robust sensors enabling condition-based maintenance of lubricated components in locomotives and wagons

Digitalization in mobility is considered the key to success to increase efficiency, reliability and safety in rail transport, both track and rolling stock. “Big data” analytics are therefore implemented. In the European Joint Undertaking “Shift2Rail”, tremendous efforts are dedicated to condition-based maintenance (CBM). The task of AC2T research GmbH is to enable data collection, processing and evaluation by appropriate sensor systems for online health status monitoring of lubricated components in locomotives and wagons to increase safety and availability while reducing of maintenance costs and unplanned downtime. Special attention is paid to robustness of both sensor and algorithm to meet the high demands for reliability in rail transport. Suitable sensor systems were compiled for three use cases at locomotives and wagons by the field-to-lab approach and validated for use in CBM.

AB0348 THE PROPER STUDY: A 48-WEEK ANALYSIS OF A PAN-EU REAL-WORLD STUDY OF SB5 BIOSIMILAR FOLLOWING TRANSITION FROM REFERENCE ADALIMUMAB IN PATIENTS WITH RHEUMATOID ARTHRITIS, AXIAL SPONDYLOARTHRITIS OR PSORIATIC ARTHRITIS

BackgroundSB5, a biosimilar to reference adalimumab (ADL), received EU marketing authorisation in 2017, based on pre-clinical and clinical phase I and III studies that demonstrated bioequivalence and comparable efficacy, safety and immunogenicity to ADL.ObjectivesThe real-world study ‘PROPER’ is designed to provide insights into outcomes of the transition from ADL to SB5 outside the randomised, controlled, clinical trial setting.MethodsUnder an umbrella design, 1000 patients with immune-mediated inflammatory disease were enrolled at centres in Belgium, Germany, Ireland, Italy, Spain and the UK, and followed for 48 weeks post-transition. Eligible patients with a diagnosis of rheumatoid arthritis (RA), axial spondyloarthritis (axSpA), psoriatic arthritis (PsA), ulcerative colitis or Crohn’s disease had been transitioned to SB5 as part of routine treatment following a minimum of 16 weeks’ treatment with ADL. Data were captured from patient charts retrospectively for 24 weeks prior to and prospectively and/or retrospectively up to 48 weeks after SB5 initiation. This analysis of the rheumatology cohort reports clinical characteristics, disease scores, persistence on SB5, clinical management and safety up to the closing date of November 30th, 2021.ResultsOf the 496 patients included in this analysis, the majority were enrolled in UK (n=174), Germany (n=145) and Spain (n=73); Italy, Ireland and Belgium enrolled 45, 44 and 15 patients respectively. At study close, 487 patients had completed 48 weeks of follow-up; 397 of those remained on SB5 throughout.Methotrexate was received as concomitant therapy by 37% of patients and 20% had received a biologic therapy prior to reference ADL. Most patients (89.3% of RA, 92.1% of axSpA, 97.3% of PsA) transitioned to SB5 at the same dose regimen received for ADL.Clinical characteristics, SB5 dose and flare are detailed in Table 1, disease scores in Figure 1.Table 1.Patient clinical characteristics, SB5 dose, flareRA (N=207)axSpA (N=127)PsA (N=162)Age at SB5 initiation (years), mean (SD); IQR60.1 (11.8)53.0, 68.050.3 (13.4)38.0, 61.053.3 (12.0)45.0, 62.0Duration of disease (years), mean (SD); IQR13.3 (11.4)5.0, 19.518.8 (13.5)9.0, 25.012.2 (9.9)4.0, 19.0n%n%n%Women15072.54031.57345.1Patients receiving SB5 40mg Q2WBaseline15273.411590.614992.0Week 4813272.59387.712491.9Episodes of Flare018790.310784.313985.81209.71814.22012.3200.021.631.9How was Flare diagnosedDisease score1155.0731.81038.5Patient-reported symptoms1995.022100.026100.0Secondary Loss of Response315.000.0726.9Action taken for FlareBiologic therapy dose adjusted420.029.1519.2Non-biologic therapy dose adjusted840.0313.6934.6Clinical investigation00.014.5311.5Other*945.01359.11661.5*Includes cessation of therapy, prescription of corticosteroids, physical exercise, no action.IQR, interquartile range; SD, standard deviation; Q2W once two-weekly.Figure 1.Disease scores (paired patients), mean (95% CI)Fifteen patients each experienced one unrelated Serious Adverse Event (SAE): 2 in the axSpA cohort [tachycardia, intracranial haemorrhage]; 6 in the PsA cohort [myocardial infarct (2), breast carcinoma, COVID-19, gallbladder calculus, dyspnoea]; 7 in the RA cohort [facial numbness, depression, COVID-19, pneumonia, diverticulitis, parvovirus, coronary occlusion]. Two patients reported SAEs considered causally related to SB5: Herpes zoster and pneumonia (RA cohort), and ALS with worsening (PsA cohort).ConclusionThis analysis of a large, contemporary cohort of EU patients with established RA, axSpA or PsA shows treatment effectiveness maintained at 48 weeks after switching from ADL to SB5, with most patients continuing on SB5 Q2W throughout. Episodes of flare were uncommon, and the importance of patient-reported symptoms in recognition of flare is evident. No new safety signals were observed.AcknowledgementsStatistical services were provided by FGK Clinical Research GmbH, Munich, Germany. Data management services were provided by Worldwide Clinical trial, Research Triangle Park, NC, USA. Funding was provided by Biogen International GmbH.Disclosure of InterestsUlf Müller-Ladner Consultant of: Biogen, Grant/research support from: Biogen, Karl Gaffney Speakers bureau: Novartis, UCB, AbbVie, Lilly, Consultant of: Novartis, UCB, AbbVie, Lilly, Pfizer, Grant/research support from: NAAS, AbbVie, Pfizer, UCB, Novartis, Lilly, Cellgene, Celltrion, Janssen, Gilead, Biogen, Deepak Jadon Consultant of: AbbVie, Celgene, Eli Lilly, Gilead, Janssen, MSD, Novartis, Oxford University Press, Pfizer, Roche, Sandoz, UCB, Grant/research support from: AbbVie, Celgene, Eli Lilly, Gilead, Janssen, MSD, Novartis, Oxford University Press, Pfizer, Roche, Sandoz, UCB, Marco Matucci-Cerinic Consultant of: Chemomab, Biogen, Pfizer, Lilly, Behring, Janssen, MSD, Eugenio Chamizo Carmona Speakers bureau: Abbvie, Amgen, Biogen, BMS, Celgene, Eli Lilly, Fresenius-Kabi, Galapagos, Janssen, MSD, Novartis, Pfizer, and UCB, Consultant of: Abbvie, Amgen, Biogen, BMS, Celgene, Eli Lilly, Fresenius-Kabi, Galapagos, Janssen, MSD, Novartis, Pfizer, and UCB, Ulrich Freudensprung Shareholder of: May hold stock in Biogen, Employee of: Biogen, Janet Addison Shareholder of: May hold stock in Biogen, Employee of: Biogen

P434 The PROPER study: interim analysis of a Pan-European real-world study of SB5 adalimumab biosimilar after transition from reference adalimumab in patients with Crohn’s disease

Abstract Background SB5, a biosimilar to reference adalimumab (ADL), received EU marketing authorisation in August, 2017, based on pre-clinical and clinical phase I and III studies that demonstrated bioequivalence and comparable efficacy, safety and immunogenicity profiles to ADL. The real-world study ‘PROPER’ is designed to provide insights into outcomes of the transition from reference to SB5 outside the controlled, randomised, clinical trial setting. Under an umbrella design, 1000 patients with immune-mediated inflammatory disease have been enrolled at centres in Belgium, Germany, Ireland, Italy, Spain and the UK. Methods Eligible patients had been transitioned to SB5 as part of routine treatment following a minimum of, 16 weeks’ treatment with ADL. Data were captured from patient charts retrospectively for, 24 weeks prior to and prospectively and/or retrospectively for, 48 weeks after SB5 initiation. This interim analysis of the Crohn’s disease cohort reports outcome measures including clinical characteristics, disease activity, persistence on SB5, clinical management and safety for patients enrolled at, 32 specialist sites and followed up to the data extract date of October, 5th, 2021. Results Of the, 462 patients included in this interim analysis, the majority were enrolled in Germany (n=127), Spain (n=118) and Italy (n=79); Belgium, Ireland and UK enrolled, 56, 46 and, 36 patients, respectively. At time of data extract, 416 patients had completed, 48 weeks of follow-up and, 15 had withdrawn from the study. The most common reasons driving transition were physician decision, or a mandate from the health authority or payer. At baseline, the majority of patients were in remission or had mild disease as determined by Harvey Bradshaw Index (HBI), and were in remission or stable according to physician opinion. Most patients transitioned to the same SB5 regimen as they had received for ADL (Table 1). Twenty-four patients reported, 27 serious adverse events (SAE), of which, 4 (anal fistula [n=1], perianal abscesses [n=2] and subileus [n=1]) were considered by the study physician to be related to SB5 administration. Conclusion No meaningful change in HBI or SB5 dosing regimen was observed from baseline to Week, 48 after transition from ADL. The number of patients experiencing a treatment-related SAE was low, and no new safety concerns were detected. Thus, transition from ADL to SB5 was generally safe and effective in patients with Crohn’s disease over, 48 weeks of follow-up. Acknowledgements: This study was funded by Biogen International GmbH. Statistical services were provided by FGK Clinical Research GmbH, Munich, Germany. Data management services were provided by Worldwide Clinical trial, Research Triangle Park, NC, USA.

Anti-CD37 Alpha-Amanitin Conjugated Antibodies As Therapeutic Weapons for Richter's Syndrome

The leukocyte surface antigen CD37 (TSPAN26), a member of the tetraspanin superfamily, is widely expressed on most malignant B cells, making it an actionable target for treatment of patients with chronic lymphocytic leukemia (CLL) and other B-cell non-Hodgkin lymphoma (NHL) indications. Accordingly, αCD37 antibodies have shown promising results in phase 1/2 clinical trials for CLL and NHL.Richter's syndrome (RS) is the transformation of CLL into an aggressive and rapidly fatal lymphoma, typically a diffuse large B cell lymphoma (DLBCL). RS is a challenging disease since very few effective treatment options exist for these patients and the available regimens, mainly based on R-CHOP scheme, show limited efficacy.We recently established patient-derived xenograft (PDX) models from RS patients and have shown that they can be used to test the efficacy of novel drugs and drug combinations 1,2. All available RS-PDX models were characterized by high-levels of CD37 expression, when assessed by RNA sequencing, reverse-transcriptase-polymerase chain reaction (RT-PCR), flow cytometry (FACS), western blot (WB) and immunohistochemistry (IHC). More precisely, two models (RS1316 and IP867/17) showed slightly higher CD37 levels compared to the others (RS9737 and RS1050).These models were used to test three different αCD37-ATACs®, ADCs which comprise amanitin-derivatives as payload. Amanitin (the main poison in the green deathcap mushroom) belongs to the well-known amatoxin family. Amanitin is taken up by OATP1B3 transporter, solely expressed on hepatocytes. Upon mushroom intoxication, it can lead to severe liver toxicity by inhibiting the RNA polymerase II. Upon conjugation to target-specific antibodies, the maximal tolerated dose is significantly increased by reducing the non-specific liver uptake. By binding to its antigen, ATACs deliver amanitin only into target-positive cancer cells while target negative cells show no off-target toxicity. Consistent with CD37 expression on the cell surface, ex-vivo treatment of freshly purified cells from RS-PDX tumor masses to αCD37-ATACs® resulted in increased apoptosis after 72 hours of treatment, with only minor differences among the 3 ATACs® and the models used. Since alpha-amanitin is a deadly toxin known to target human RNA polymerase II and, at high doses, also RNA polymerase III, we checked messenger RNA levels in basal conditions and after CD37-ATAC® treatment by looking at different housekeeping genes, and confirmed a reduction in global mRNA levels.αCD37-ATAC® efficacy was then assessed in vivo in systemic RS-PDX models where RS cells are intra-venously (i.v.) injected in the tail vein and cells distribute to different tissues (blood, spleen and bone marrow), resembling the human disease. Cells from RS1316, RS1050 and RS9737 models were injected into the tail vein and left to engraft 14 days, before mice were randomly assigned to vehicle or ATAC® groups. A single i.v. treatment for each αCD37-ATAC® was administered, testing two different doses for each compound, and mice were then monitored for survival. Overall, the single administration of all three ATACs® caused highly significant disease regression. In the RS1316 model, independently of the dose or tested ATAC®s, all treated mice, except one, were alive and disease-free until the end of the experiment, 140 days post cells injection, while survival of vehicle-treated mice was 65 days. FACS analysis to trace neoplastic cells in parenchymatous organs and bone marrow confirmed the absence of neoplastic cells. In the other 2 models, RS9737 and RS1050, even though ATAC® treatment did not result in complete disease eradication, a single administration of αCD37-ATAC® resulted in a dramatically increased survival (approximately 35-60 days, depending on the model and ATAC® used).Finally, CD37 expression was confirmed by RNA sequencing on a cohort of 15 primary RS samples, even though with variable levels. Compared to CLL cells, RS samples showed CD37 expression levels comparable to those reported for DLBCL cells.Overall, these data indicate CD37 as a potential target to treat RS patients with selective targeting αCD37-ATACs®.ATACs® is a registered trade mark of Heidelberg Pharma Research GmbH, Germany

CHEM Research GmbH: Decline in German Paint and Coating Consumption in Q1 2021

CHEM Research GmbH: Decline in German Paint and Coating Consumption in Q1 2021

Preclinical Characterization of AMG 424, a Novel Humanized T Cell-Recruiting Bispecific Anti-CD3/CD38 Antibody

Introduction: Multiple myeloma (MM) is a highly prevalent hematological malignancy, with a worldwide incidence of over 62,000 patients in 2012. Despite significant progress in the development of MM treatments in the last decade, there is a great unmet medical need for MM patients who relapse on existing therapies. Given the greater time needed for relapses to manifest when minimal residual disease (MRD) is reduced, new therapies should be aimed at eliminating MRD. Bispecific T cell engager (BiTE®) molecules recruit T cells to cancer cells and trigger T cell-dependent cellular cytotoxicity (TDCC). BiTEs are clinically validated for the treatment of hematological malignancies and have the potential to greatly reduce MRD. CD38 is a membrane protein normally expressed by plasma cells, lymphocytes, and other immune cells and is a clinically validated tumor-associated antigen expressed at high levels on MM cells. Herein, we describe for the first time AMG 424, a novel, humanized T cell-recruiting bispecific anti-CD3/CD38 antibody containing an XmAb® Fc domain that cross-reacts with nonhuman primate (NHP) CD3 and CD38. AMG 424 was selected for its affinity for CD3 and CD38 in an effort to overcome the challenges associated with CD38 and CD3 binding.Methods: A panel of eight novel bispecific molecules that bind with various affinities to CD3 and CD38 were tested in a series of in vitro assays, including TDCC against cancer cell lines, autologous B cell depletion, cytokine release, and soluble CD38 interference assays. AMG 424 was selected from this panel and evaluated in vivo for its ability to induce tumor growth inhibition and prolong survival in an orthotopic MOLM-13-luc xenograft model in NOD/SCID gamma mice co-implanted with human T cells. AMG 424 was also tested in a repeat dose study in NHPs in which depletion of peripheral CD38-expressing target cells was monitored as a pharmacodynamic endpoint. Finally, the potential of AMG 424 to be combined with relevant standard-of-care therapies was evaluated in vitro.Results: In vitro, AMG 424 demonstrated complete redirected lysis activity against cancer cells with CD38 surface density as low as ~2,000 copies per cell. Compared with molecules with greater CD3 affinity, AMG 424 triggered the release of less IFN-γ and TNF-α in the presence of target cells. Moreover, in the presence of soluble CD38 (200 ng/mL), the in vitro potency of AMG 424 was decreased less than two-fold, whereas molecules with greater affinity for CD38 were decreased nearly ten-fold. Thus, the reduced affinity of AMG 424 for CD3 and CD38 versus other molecules may improve tolerability and pharmacokinetic variability. In tumor-bearing mice with pre-activated human T cells, AMG 424 induced 98% tumor growth inhibition and prolonged survival by more than 70%. In NHPs, AMG 424 depleted peripheral CD38-expressing target cells. In vitro, AMG 424 activity was minimally affected when combined with existing standard-of-care therapies.Conclusions: AMG 424 is a novel, humanized, bispecific T cell-recruiting anti-CD3/CD38 antibody that targets MM cancer cells through T-cell dependent redirected lysis, an approach with the potential to eliminate MRD. AMG 424 completely cleared target cells in TDCC assays and triggered depletion of target cells in vivo. These results support further investigation of AMG 424 as a single agent or combined with existing standard-of-care therapies. Disclosuresde Zafra:Amgen Inc.: Employment. Balazs:Amgen Inc.: Employment, Equity Ownership. Fajardo:Amgen Inc.: Employment, Equity Ownership. Liang:Amgen Inc.: Employment, Equity Ownership. Zhong:Amgen Inc.: Employment, Equity Ownership. Henn:Amgen Research (Munich) GmbH: Employment, Equity Ownership. Bernett:Xencor, Inc.: Employment, Equity Ownership. Moore:Xencor, Inc.: Employment, Equity Ownership. Muchhal:Xencor, Inc.: Employment. Winters:Amgen Inc.: Employment, Equity Ownership. Frank:Amgen Inc.: Employment, Equity Ownership. Cook:Amgen Inc.: Employment, Equity Ownership. Pearson:Amgen Inc.: Employment, Equity Ownership. Melhem:Amgen Inc.: Employment. Petrovic:Amgen Inc.: Employment, Equity Ownership. Pelham:Amgen Inc.: Employment, Equity Ownership. Friedrich:Amgen Research (Munich) GmbH: Employment, Equity Ownership. Tan:Amgen Inc.: Employment, Equity Ownership. Moody:MedImmune, Inc.: Employment; Amgen Inc.: Equity Ownership. Stevens:Amgen Inc.: Employment, Patents & Royalties. Desjarlais:Xencor, Inc.: Employment. Coxon:Amgen Inc.: Employment, Equity Ownership. Nolan-Stevaux:Amgen Inc.: Employment, Equity Ownership.

Evaluation of a FLT3 Bite® for Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a disease with high unmet medical need. Standard induction chemotherapy can lead to complete remission in patients with a favorable genetic risk profile; however, about half of patients are ineligible for this intensive regimen. In those patients with a non-favorable genetic risk profile, the preferred treatment strategy is allogeneic stem cell transfer, but the majority of patients are ineligible due to age and other comorbidities.FLT3 is a receptor tyrosine kinase that induces cell growth in myeloid progenitor cells. Activating mutations in FLT3 have been identified in approximately 30% of AML patients. Expression of mutant FLT3 and overexpression of wild type FLT3 are associated with poor prognosis in AML, leading to clinical investigation of targeted FLT3 inhibitors, including midostaurin which was recently approved for patients with newly diagnosed FLT3-mutant AML. These inhibitors are likely to be effective only in certain patient subpopulations and may ultimately give rise to acquired resistance. To drive cures in AML, effective therapies for broad patient populations that can eliminate residual disease are needed.Bispecific T cell engaging antibody constructs (BiTE®) are a class of immunotherapy agents which redirect T lymphocytes to a specific antigen expressed on tumor cells. Blinatumomab, a BiTE® containing a single chain variable fragment (scFv) that binds CD19 on B lymphocytes and an scFv that binds CD3 on T lymphocytes, has shown clinical efficacy for treating CD19-positive hematological malignancies such as ALL. To extend the serum half-life, next generation BiTE® antibody constructs have been modified to contain Fc-moieties, allowing for weekly dosing.To evaluate FLT3 as a BiTE® target in AML, we assessed expression of FLT3 on primary AML patient blasts using flow cytometry. Over 83% of AML patients (n = 192) were FLT3-positive (MFI ratio > 1.5) with similar expression in bulk and leukemic stem cells. A half-life extended FLT3 BiTE® (FLT3 HLE BITE®) was generated and evaluated in vitro as well as in vivo in a mouse xenograft tumor model and non-human primates (NHP). FLT3 HLE BiTE® had subnanomolar binding affinity for human FLT3 and nanomolar binding affinity for human CD3 and NHP FLT3 and CD3. In cytotoxic assays targeting human AML cell lines, FLT3 HLE BiTE® exhibited picomolar efficacy using as effector cells human pan T cells in a 10:1 E:T ratio (range: 2 to 10 pM) or NHP PBMCs in a 5:1 E:T ratio (range: 30 to 80 pM). In mice injected with FLT3-positive MOLM13 cells and human CD3-positive T cells, FLT3 HLE BiTE® induced robust anti-tumor activity and extended survival ≥ 2-fold at all doses (n = 10 per group, p ≤ 0.0015). Following a single 5 µg/kg dose in NHP (n=2), FLT3 HLE BiTE® exhibited a reproducible pharmacokinetic profile with a 66 hour half-life. In this study, we observed hallmarks of BiTE® activation such as induction of IL-6 and MCP-1 and indication of a pharmacodynamic effect through increased soluble FLT3 ligand. Following repeat-dosing with FLT3 HLE BiTE®, digital droplet PCR was used to measure FLT3 transcript in NHP blood and bone marrow. Robust depletion of FLT3+ cells was observed in both NHP blood (≥ 95% reduction) and bone marrow (≥ 68% reduction) at all doses (5 µg/kg, 25 µg/kg, 75 µg/kg, n = 3/group). Taken together, FLT3 HLE BiTE® has potent in vitro and in vivo activity and may provide therapeutic benefit to AML patients. DisclosuresGoldstein:Amgen: Employment. Henn:Amgen Research (Munich) GmbH: Employment, Equity Ownership. Archibeque:Amgen: Employment. Frank:Amgen Inc.: Employment, Equity Ownership. Balazs:Amgen Inc.: Employment, Equity Ownership. Dahlhoff:Amgen Research (Munich): Employment, Equity Ownership. Raum:Amgen Research (Munich): Employment. Li:Amgen, Inc.: Employment. Wahl:Amgen Research (Munich): Employment. Rock:Amgen, Inc.: Employment, Equity Ownership. Thomas:Amgen Research (Munich): Employment. Karbowski:Amgen, Inc.: Employment. Krupka:Amgen Research (Munich): Research Funding; Amgen, Inc: Research Funding. Subklewe:Amgen Research (Munich): Research Funding; Amgen, Inc.: Research Funding. Coxon:Amgen, Inc.: Employment. Chapman-Arvedson:Amgen, Inc.: Employment, Equity Ownership.

Generation of a Half-Life Extended Anti-CD19 BiTE® Antibody Construct Compatible with Once-Weekly Dosing for Treatment of CD19-Positive Malignancies

CD19 is an immunoglobulin family member expressed ubiquitously on B cell lineage cells, from the pre-B cell to the mature B cell stage, until downregulation during terminal differentiation into plasma cells. CD19 is important for B cell activation, maturation and proliferation, and is widely expressed on the majority of B cell neoplasms.The T cell engaging bispecific antibody construct (BiTE®) blinatumomab, which targets CD19, is approved for the treatment of relapsed or refractory B cell precursor acute lymphoblastic leukemia in adults and children in the US and has shown great promise for treating other CD19-positive hematological malignancies. The short serum half-life of blinatumomab necessitates administration by continuous IV infusion to maintain therapeutic serum concentrations. To extend the half-life and allow for a more convenient administration, a next generation BiTE® antibody construct designated CD19 HLE BiTE® has been generated. The CD19 HLE BiTE® comprises a new CD19- and CD3-targeting tandem single-chain Fv antibody fused to an Fc domain.The biological properties of CD19 HLE BiTE® were evaluated in vitro and in vivo . CD19 HLE BiTE® binds with high affinity to both human and non-human primate (NHP) CD19 as well as CD3. In vitro experiments demonstrated that CD19 HLE BiTE® can recruit and activate human and NHP T cells, mediating redirected lysis of CD19-expressing cells at half-maximal effective concentrations in the low picomolar range. The fusion to an Fc domain resulted in a serum half-life of 210 hours following a single intravenous administration of a 5 µg/kg dose in NHP. In a repeat-dose study CD19 HLE BiTE® caused robust depletion of circulating B cells, B lineage bone marrow cells, and peripheral lymphoid organ B cells in NHPs, whilst no overt signs of toxicity were observed in clinical and laboratory examinations of the animals.These results demonstrate that CD19 HLE BiTE® effectively eliminated CD19-positive cells in vitro and in vivo, and may be suitable for once weekly dosing in patients with CD19-positive malignancies. DisclosuresLorenczewski:Amgen: Employment, Equity Ownership. Friedrich:Amgen Research (Munich) GmbH: Employment, Equity Ownership. Kischel:AMGEN: Employment. Dahlhoff:Amgen Research (Munich): Employment, Equity Ownership. Anlahr:Amgen: Employment, Equity Ownership. Balazs:Amgen Inc.: Employment, Equity Ownership. Rock:Amgen, Inc.: Employment, Equity Ownership. Boyle:Amgen: Employment. Goldstein:Amgen: Employment. Coxon:Amgen, Inc.: Employment. Chapman-Arvedson:Amgen, Inc.: Employment, Equity Ownership.

AB0204 THE PROPER STUDY: INTERIM ANALYSIS OF A PAN-EU REAL-WORLD STUDY OF SB5 BIOSIMILAR FOLLOWING TRANSITION FROM REFERENCE ADALIMUMAB IN PATIENTS WITH RHEUMATOID ARTHRITIS, AXIAL SPONDYLOARTHRITIS, OR PSORIATIC ARTHRITIS

Background:SB5, a biosimilar to reference adalimumab (ADL), received EU marketing authorisation in August 2017, based on the totality of evidence from pre-clinical and clinical Phase I and III studies that demonstrated bioequivalence, similar efficacy, and comparable safety and immunogenicity to the reference. This real-world study provides data on outcomes of the transition from reference to biosimilar ADL outside the controlled, randomised, clinical trial setting.Objectives:To evaluate candidate predictors of persistence on SB5 in EU patients (pts) across multiple indications.Methods:This ongoing observational study enrolled 1000 pts with rheumatoid arthritis (RA), axial spondyloarthritis (axSpA), psoriatic arthritis (PsA), ulcerative colitis, or Crohn’s disease who initiated SB5 as part of routine clinical practice following a minimum of 16 weeks’ treatment with reference ADL, at clinics in Belgium, Germany, Ireland, Italy, Spain, and the UK. Data are captured from clinic records retrospectively for the 24 weeks prior to transition, and prospectively and/or retrospectively for 48 weeks following transition. Primary outcome measures include baseline clinical characteristics, disease activity scores and clinical management over time; data on COVID-19 infection has recently been captured. This interim analysis (IA) provides an overview of baseline characteristics, disease scores and dose regimen up to 48 weeks post-initiation of SB5, and COVID-19 infection reported to date, in subjects with RA, axSpA, or PsA enrolled at 35 specialist sites and followed up to the data extract date of 18th December 2020.Results:Of the 504 pts included in this IA, 201 have RA, 169 have PsA, and 134 axSpA. At time of data extract, 216 pts had completed 48 weeks on SB5, 73 pts had discontinued SB5, and 8 had discontinued the study.RA (N=201)axSpA (N=134)PsA (N=169)Mean (SD)Q1, Q3Mean (SD)Q1, Q3Mean (SD)Q1, Q3Age at SB5 initiation (years)60.2 (11.7)53, 6850.5 (13.6)38, 6153.0 (12.2)43, 62Duration of disease (years)13.5 (11.7)4.5, 2018.7 (13.2)9, 2512.7 (9.9)4, 20n%n%n%Women14471.64130.67745.6SB5 Dosing regimen:Baseline 40 mg Q2W14974.112089.615692.3Week 48 40mg Q2W6977.56785.99892.5Baseline Other*5225.91410.4137.7Week 48 Other*2022.41114.187.5Disease Score(paired patients)DAS28FFbHBASDAITender Joint CountSwollen Joint CountBaseline, n, mean (95% CI)692.5 (2.3–2.7)2273.9 (65.6–82.1)422.8 (2.3–3.4)491.8 (0.1–3.0)490.6 (0.2–0.9)Week 48, n, mean (95% CI)692.6 (2.3–2.8)2272.1 (64.0–80.2)423.0 (2.4–3.7)491.9 (0.5–3.3)490.6 (0.1–1.1)Patient diagnosed with COVID-19 at any time on-study, n (%)No14295.39688.114098.6Yes32.010.910.7Unknown42.71211.010.7Imraldi regimen stopped or changed due to COVID-19, regardless of diagnosis, n (%)No13098.510697.2134100Yes21.532.800SD standard deviation; Q1 1st quartile, Q3 3rd quartile; CI Confidence Interval*Other includes all other reported doses and/or dosing intervals: 40mg QW, 80mg Q2W, and unspecified frequencyDAS28 Disease Activity Score 28; FFbH Hanover Functional Ability Questionnaire; BASDAI Bath Ankylosing Spondylitis Disease Activity IndexConclusion:This IA provides a first insight into clinical management of pts over 48 weeks, in a contemporary cohort of EU pts with established RA, axSpA and PsA, switched from reference to biosimilar ADL SB5 in clinical practice. The majority of pts showed no meaningful difference in disease score or dose regimen of SB5 by Week 48 post-transition. As of data extract date, the proportion of pts with a known positive COVID-19 test was low (1.3%) and a small minority (1.3%) had SB5 treatment changed or interrupted as a result of the COVID-19 pandemic. With follow-up of pts ongoing to Q4 2021, the study will continue to provide pertinent information about clinical outcomes of transition from reference to biosimilar ADL in real-world practice and in indications not investigated in controlled studies.Acknowledgements:Statistical services gave been provided by FGK Clinical Research GmbH, Munich, Germany. Data management services were provided by Worldwide Clinical trial, Research Triangle Park, NC, USA; funding was provided by Biogen International GmbH.Disclosure of Interests:Ulf Müller-Ladner Consultant of: Biogen, Grant/research support from: Biogen, Karl Gaffney Consultant of: AbbVie, Celgene, Gilead, Lilly, MSD, Novartis, Pfizer, and UCB, Grant/research support from: AbbVie, Celgene, Gilead, Lilly, MSD, Novartis, Pfizer, and UCB, Deepak Jadon Consultant of: AbbVie, Celgene, Eli Lilly, Gilead, Janssen, MSD, Novartis, Oxford University Press, Pfizer, Roche, Sandoz, UCB, Grant/research support from: AbbVie, Celgene, Eli Lilly, Gilead, Janssen, MSD, Novartis, Oxford University Press, Pfizer, Roche, Sandoz, UCB, Ulrich Freudensprung Shareholder of: Biogen, Employee of: Biogen, Janet Addison Shareholder of: Biogen, Employee of: Biogen

Predictors of Efficacy for Blinatumomab in BCP-ALL Patients: Non-Responders Show Impaired CD19-BiTE®-Mediated Cytotoxicity in Vitro

Bispecific T-cell engaging (BiTE®) antibody constructs recruit T cells to target antigens independent of their T-cell receptor specificity. Blinatumomab, a CD19xCD3 BiTE® antibody construct, is approved for the treatment of relapsed/refractory (r/r) B-cell precursor acute lymphoblastic leukemia (BCP-ALL), including patients with persistence or reoccurrence of measurable residual disease (MRD). Despite superior anti-leukemic efficacy compared to cytostatic agents, a majority of patients do not respond to treatment. Biomarkers for the identification of non-responders prior to or early during treatment are unknown. However, the definition of robust biomarkers for efficacy is of high importance for clinical decision-making and might also indicate ways to improve response rates. We therefore established a comprehensive immune-monitoring program for blinatumomab patients. We sequentially analysed peripheral blood of patients with r/r or MRD-positive disease receiving blinatumomab prior to the start of infusion and once weekly over the course of the first 28-day cycle. We determined CD3+ T cell counts and their subset distribution (CD4, CD8, naïve [TN], central memory [TCM], effector memory [TEM], and effector memory CD45RA-positive [TEMRA] T cells) by multiparameter flow cytometry (MPFC). Additionally, patient-derived T cells were cocultered with BCP-ALL cell lines (SEM and REH) at an effector: target ratio of 1:3 in presence of blinatumomab or a control BiTE® (0.5 ng/ml, respectively) for 3 days to assess their cytotoxic capacity. Blinatumomab-mediated cytotoxicity was determined by MPFC. T-cell proliferation was evaluated by MPFC (Far Red Cell tracer) after incubation with CD3/CD28 beads for 6 days. A total of 16 patients were enrolled. Four of these patients suffered from MRD disease, whereas the remaining 12 had overt relapse of BCP-ALL. Response rates for patients with morphological relapse were 50% (4 CRMRD- and 2 CRMRD+ after the first cycle) and 75% for patients with MRD disease (3 MRD conversions, 1 MRD persistence). Absolute lymphocyte counts were not significantly different between responders and non-responders before treatment initiation (0.9 G/l and 0.7 G/l, respectively). Whereas the percentage of CD3+ T cells (of all lymphocytes) did not significantly differ between responders and non-responders on day 0, non-responders had a significantly reduced CD3+ percentage on day 7 (81.2% vs 92.9%, p=0.03). Until day 28 of the first cycle, CD3+ percentages of responders and non-responders re-converged (80.2% and 81.4%, respectively). There were no significant differences for CD4+ and CD8+ T-cell percentages prior to and over the course of the first cycle. This was also true for TN, TCM, TEM and TEMRA subset distributions. Additionally, CD19-BiTE®-mediated cytotoxicity of patient-derived T cells was assessed against CD19 expressing target B cell lines in vitro. Interestingly, specific lysis did not differ between responders and non-responders on day 0 (89.6% vs 79.8%, p=0.89), but decreased for non-responders over the course of the first cycle (normalized AUC for cytotoxicity 149 vs 403 in responders, p=0.03). However, this observation was only true for the patients with refractory morphologic relapse, as the non-responding MRD patient maintained T cell cytotoxicity over the first cycle of therapy. Impaired T cell proliferation (leading to reduced E:T ratios) might contribute to the observed dysfunctional cytotoxicity in non-responders, as the percentage of proliferating T cells after CD3/CD28 bead-based stimulation tended to be reduced for non-responders (17.9% vs 36.1%, p=0.07). In summary, lymphocyte counts, T cell percentage and T cell subset distributions do not allow for a response prediction prior to treatment start for patients receiving blinatumomab. BCP-ALL patients with morphological relapse/persistence who do not achieve a remission with blinatumomab therapy show reduced cytotoxicity in vitro in comparison to patients responding to treatment. As this observation could not be confirmed with one MRD non-responder in our cohort, the burden of disease might contribute to the observed T cell dysfunction, possibly by interfering with T cell proliferation. Evaluation of immune checkpoint expression on effector and target cells over the course of the therapy is currently ongoing, as are analyses of the T cell transcriptome of responders and non-responders. Disclosures Kischel: AMGEN Research (Munich) GmbH: Employment, Equity Ownership. Subklewe:Celgene: Consultancy, Honoraria; Oxford Biotherapeutics: Research Funding; Miltenyi: Research Funding; Roche: Consultancy, Research Funding; Janssen: Consultancy; Gilead: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Morphosys: Research Funding; AMGEN: Consultancy, Honoraria, Research Funding.

Impact of p53 Knock-Down on T-Cell Proliferation and T-Cell Mediated Cytotoxicity Against AML Cell Lines Mediated By a CD33 Specific BiTE® Antibody Construct

Impact of p53 Knock-Down on T-Cell Proliferation and T-Cell Mediated Cytotoxicity Against AML Cell Lines Mediated By a CD33 Specific BiTE® Antibody Construct

AMG 701 Potently Induces Anti-Multiple Myeloma (MM) Functions of T Cells and IMiDs Further Enhance Its Efficacy to Prevent MM Relapse In Vivo

AMG 701 is a half-life extended BiTE® (bispecific T-cell engager) targeting the B cell maturation antigen (BCMA). Here, we confirmed AMG 701-mediated T cell-redirected lysis of MM cells, defined immunomodulatory effects of AMG 701, and investigated combination potential of AMG 701 with immunomodulatory drugs (IMiDs) in human MM. Firstly, AMG 701 induced specific and efficacious T cell-dependent cytotoxicity (TDCC) against all MM cell lines tested, regardless of sensitivity to current anti-MM agents and expression levels of BCMA. AMG 701-induced TDCC was minimally affected in the presence of myeloma-supporting cells and cytokines in the bone marrow (BM) microenvironment, including osteoclasts (OCs), BM stromal cells (BMSCs), and a proliferation-inducing ligand (100 ng/ml). Importantly, AMG 701 induced lysis of autologous patient cells from the relapse and refractory stage of MM (RRMM). AMG 701 rapidly upregulated cell surface expression of CD107a and the production of IFNγ and TNFα, more so in CD8 than CD4 T subsets. It stimulated the proliferation and activation of T cells, to a greater extent in CD8 vs CD4 T cells, leading to significantly increased ratios of CD8/CD4 T cells. Significantly, AMG 701 induced differentiation of naive T cells (CD4 and CD8) to T cells with memory phenotype. This includes central memory (CM), effector memory (EM) T cells, and stem cell like memory cells. Time-course immunophenotyping studies showed that AMG 701 transiently upregulated the expression of key immune checkpoint and costimulatory markers on both CD4 and CD8 T cells. The induced T cells purified from ex vivo co-cultures still effectively lysed MM cells with lower BCMA levels. This may suggest an increased T cell clonality. Furthermore, IMiDs (len or pom) enhanced AMG 701-mediated TDCC against MM cells at earlier time points, lower E/T ratios, lower concentrations, or in the presence of immunosuppressive OCs or BMSCs. The combination AMG 701 and IMiDs maximized MM cell lysis accompanied with a decreased EC50 value. Combined treatments induce a more pronounced immunomodulation than AMG 701 alone in the presence of OCs, as evidenced by higher percentage of CM+EM and CD8/CD4 ratio at d8. AMG 701 with IMiDs combination significantly enhance AMG 701-mediated autologous patient MM cell lysis in a synergistic manner (combination index < 1). In the human NCI-H929 xenograft model reconstituted with human T effector cells, AMG 701 effectively blocked tumor growth 5d after the first injection, regardless of doses (0.02-2 mg/kg). Tumors were completely eradicated following 3 separate injections in the host without weight loss. Next, sub-optimal doses and treatment schedules for AMG 701 and len were then used to investigate in vivo anti-MM effects by the combination vs monotherapy. Mice receiving MM cells were treated, from d15 until the end of the study, with len once daily, AMG 701 once weekly, or combination of AMG 701 and len. Two days after the first drug administration, all three treatments significantly inhibited MM tumor growth in mice (p<0.001). Most importantly, while AMG 701 or len group showed tumor progress eventually, the combination of AMG 701 with len continuously suppressed tumor growth (p<0.05 after d26; p<0.001 after d40 for combination vs either agent alone). Combination of AMG 701 and len significantly induced superior MM cell regression, compared to either monotherapy, resulting in enhanced tumor regression and prevention of disease relapse. Taken together, these results strongly support AMG 701-based clinical studies, both as monotherapy (NCT03287908) and in combination with IMiDs to enhance elimination of residual diseases and prolong long-term durable responses in MM. Disclosures Munshi: Oncopep: Consultancy; Janssen: Consultancy; Abbvie: Consultancy; Takeda: Consultancy; Adaptive: Consultancy; Amgen: Consultancy; Celgene: Consultancy. Wahl:Amgen Research GmbH: Employment. Matthes:Amgen Research GmbH: Employment. Anderson:Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau. Chapman-Arvedson:Amgen Research: Employment.

Practical Applications of The Predictability of Alternative UCITS Fund Returns

Practical Applications Summary In The Predictability of Alternative UCITS Fund Returns, from the Summer 2019 issue of The Journal of Alternative Investments, authors Michael Busack (Absolut Research GmbH), Wolfgang Drobetz (Hamburg University), and Jan Tille (Absolut Research) ask if it is possible to predict the future performance of European mutual funds known as “alternative UCITS”—publicly available funds that use nontraditional investment strategies like those of private hedge funds. Alternative UCITS managers can change their investment and trading strategies based on predictions of how the market and the economy will perform. The authors decided to find out whether individual investors can also predict how well individual alternative UCITS funds will perform. The answer is yes—if individual investors have access to sophisticated statistical models. Employing 13 variables that are commonly used to predict the performance of the market and the economy, the authors selected alternative UCITS that statistical models said would constitute the top 10% of performers. Those funds produced significantly higher earnings than the historical average, with the greatest benefits occurring during market crises. TOPICS:Exchange-traded funds and applications, real assets/alternative investments/private equity, performance measurement

Secreted Mutant Calreticulins As Rogue Cytokines Trigger Thrombopoietin Receptor Activation Specifically in CALR Mutated Cells: Perspectives for MPN Therapy

BackgroundMutant calreticulins carrying the sequence translated after a +1 frameshift at the C-terminus are major drivers of myeloproliferative neoplasms (MPNs). These mutant CALRs bind and activate TpoR/MPL in cells co-expressing TpoR and mutant CALRs, resulting in persistent JAK2-STAT5 signaling. Whether mutant CALR proteins are secreted, thus acting in trans on other cells, is not known.AimsOur objectives were to: 1) assess the direct TpoR-mutant CALR interaction both when expressed in the same or in different cells; 2) determine whether mutant CALRs are secreted; and 3) determine whether mutant CALR can act as extracellular cytokines.MethodsEngineered CALR and TpoR mutants were analyzed by a combination of biochemical approaches (bioluminescence resonance energy transfer, recombinant protein production), functional assays (cell growth and transcriptional assays, flow cytometry, primary megakaryocytic clonogenic assay, analysis of CALR del52 knock-in mice) and cell imaging (confocal microscopy, flow cytometry and immuno-gold electron microscopy). Secreted CALRs were determined by ELISA using mutant specific antibodies.Results1) Two systems provided evidence that mutant CALRs and TpoR directly interact. First, using Nano-BRET in cells co-expressing N-terminally fused TpoR or EpoR with Nano-luciferase and mutant or WT CALR C-terminally tagged with HaloTag that is bound to the 618-ligand fluorophore, we show that TpoR and mutant CALRs interact in a complex whether the two proteins are within 10 nm. The interaction does not occur between TpoR and WT CALR, or between EpoR and mutant or WT CALRs. Second, expressing mutant CALR and TpoR extracellular domain in S2 Drosophila Schneider cells showed that stable complex formation requires immature high mannose structure on TpoR. Lastly, we could detect surface expression of the TpoR/CALRdel52 complex using proximity ligation assay with anti-TpoR and anti-mutant CALR antibodies in CRISPR/Cas9 engineered UT7/Tpo cells that express endogenous mutant CALR and TpoR levels.2) We used flow cytometry, confocal immunofluorescence and immunogold electron microscopy and could show that mutant CALRs are trafficking via cis-, medial- and trans-Golgi to the cell-surface and are secreted, independently from TpoR expression. Importantly, mutant CALRs are also secreted in CALR mutated MPN patients as determined by mutant CALR-specific ELISA assay in patient plasma (mean plasma level 24.6 ng/ml, range 0-156.5 ng/ml). In the 113 evaluated CALR mutated patients from different centers the plasma mutant CALR levels correlated with CALR mutant allele burden (P<0.001). Secreted mutant CALR can also be found in plasma from knock-in CALR del52 mice.3) We show that recombinant mutant CALR can act as a cytokine and specifically stimulate JAK2-STAT5 pathway in cells that carry the TpoR at the surface. Using Nano-BRET, we could demonstrate that extracellular mutant Halo-tagged CALR can specifically bind in trans to the cell-surface TpoR fused with Nano-luciferase, but not to EpoR fused with Nano-luciferase. This binding and the subsequent JAK2 activation were obtained at levels of around 100-150 ng/ml only in cells exposing at the cell-surface TpoR with at least one immature N-linked sugar. This can be accomplished by co-expressing in the reporter cells non-tagged mutant CALR, which will promote cell-surface localization of partially immature TpoR. The effect of exogenous mutant CALR could involve both stabilization of the endogenous cell-surface mutant CALR-TpoR complexes and binding to unoccupied immature TpoRs.ConclusionWe show that mutant CALRs directly interact with TpoR and also are secreted and can act as rogue cytokines, leading to activation of cells carrying TpoR. Activation of TpoR in trans is efficient at mutant CALR levels similar to those detected in patients when target cells co-express heterozygous mutant CALR and TpoR, where endogenous mutant CALR transports to the surface TpoR with immature glycosylation. Thus, secreted mutant CALRs is predicted to expand the MPN clone. Given that cell-surface mutant CALR in TpoR expressing cells is crucial for oncogenicity, and that mutant CALRs are also secreted correlating with allele burden, we discuss how antibodies and other immunotherapy approaches could specifically target the mutant CALR MPN clone. DisclosuresXu:MyeloPro Research and Diagnostics GmBH: Employment. Hug:MyeloPro Diagnostics and Research GmbH: Employment. Gisslinger:Janssen: Consultancy, Honoraria; AOP Orphan: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Shire: Honoraria; Novartis: Consultancy, Honoraria, Research Funding. Kralovics:MyeloPro Diagnostics and Research GmbH: Equity Ownership. Constantinescu:Personal Genetics: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Novartis: Membership on an entity's Board of Directors or advisory committees; AlsaTECH: Equity Ownership; Novartis: Honoraria; MyeloPro Research and Diagnostics GmbH: Equity Ownership.

Mutational Landscape of the Transcriptome Offers a Rich Neoantigen Resource for Immunotherapy of Myeloproliferative Neoplasms

BackgroundMyeloproliferative Neoplasms (MPN) encompass several disease subgroups (ET, PV, and PMF) with distinct phenotypic and clinical features. Although the landscape of somatic mutations in MPN has been mapped in detail, the current therapies show limited efficacy in eliminating MPN cells. Immunotherapy for MPN have been suggested as an alternative to standard therapies, however, a broader view on the neoantigen landscape and MHC class I (MHCI) restriction of putative MPN neoantigens is missing.AimsWe aimed to map the mutation and neoantigen landscape of MPN by analyzing whole transcriptome data and examine the applicability of targeted immunotherapy by analyzing co-occurrence of specific neoantigens with high affinity MHCI molecules in individual patients.MethodsWe have performed whole transcriptome sequencing of granulocytes for 113 MPN patients (32 ET, 17 PV, 55 PMF, and 9 post-MPN AML) and 15 controls. We implemented customized and established algorithms for genome wide fusion gene discovery, variant calling on 87 myeloid relevant genes, detection of splicing abnormalities and inferred these mutation classes directly from RNA-seq data for each patient. We extracted HLA genotypes from the same RNA-seq dataset. We examined the impact on protein sequence for each mutation class and predicted peptide affinities to MHCI. Top predicted peptide:MHCI interactions were validated in vitro using the UV-induced peptide exchange method.ResultsFollowing data processing and filtering, we identified a total of 13 gene fusions, 231 non-synonymous SNVs, and 21 Indels in 106/113 patients. Fusions were validated with Sanger sequencing, for SNVs and Indels, we re-sequenced DNA of 77 patients using a TruSight targeted sequencing panel. An unexpected high frequency of SF3B1 (0% ET, 14.6% PMF, 0% PV) hotspot mutations (K666N/R/T, K700E) was identified. We performed differential splice junction analysis comparing SF3B1 mutated and non-mutated patients and found 895 significantly differentially spliced junctions (750/895 were mediated through alternative 3'splicing). For each junction we calculated the percent splice in value (PSI) expressing the relative abundance of the alternative junction. We identified 20 junctions with retained intronic sequences with PSI>20%. For each of the 20 splicing defects we evaluated the impact of intron retention on the protein sequence. These splicing defects caused insertion of novel amino acids either in frame or through frameshifts. From all the mutations detected, we generated a virtual peptide library. In 113 patients, we detected 541 patient specific peptides predicted to bind patients' own MHCI proteins of which 102 had a predicted strong and 439 weak binding affinity. The recurrent CALR frameshifts and MPL-W515K/L/A mutations were also a rich resource for potential neoantigens due to their unique property to bind many shared common MHCI molecules (42, and 17 predicted for CALR and MPL, respectively). SF3B1 mutated patients had the highest number of predicted recurrent neoantigens with an average of 38 weak and 16 strong peptide/MHCI pairs. In total, we could identify 149 unique neoantigens covering 62% of MPN patients as a potential target in personalized cancer immunotherapy approaches. Of the 149 predicted unique neoantigens we tested 35 top ranking peptides for binding to recombinant MHCI monomers. We validated 70% of these peptides to be strong MHCI binders.ConclusionsOur results provide a framework for systematic mining of neoantigens from different mutation classes using RNA analysis. The data presented in this study may serve as a resource for development of personalized vaccines or adoptive cell-base therapies, in particular for PMF patients positive for CALR, MPL and SF3B1 mutations. DisclosuresRosebrock:MyeloPro Diagnostics and Research GmbH: Employment. Hug:MyeloPro Diagnostics and Research GmbH: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Stengel:MLL Munich Leukemia Laboratory: Employment. Gisslinger:Takeda: Consultancy, Honoraria; AOP Orphan: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria; Shire: Honoraria. Kralovics:MyeloPro Diagnostics and Research GmbH: Equity Ownership.

Mebendazole Exerts Potent Anti-Leukemic Effects By Downregulating Protein Levels of Hedgehog Transcription Factors GLI1 and GLI2

The relevance of the Hedgehog signaling pathway in the pathophysiology of acute myeloid leukemia (AML) has been demonstrated by us and others. Inhibition of the downstream Hedgehog transcription factors GLI1 and GLI2 results in strong anti-leukemic effects. Therefore, Hedgehog pathway inhibitors represent a promising therapeutic approach in AML. Mebendazole is an anthelmintic drug commonly used for the treatment of various parasitic worm infections. Recently, mebendazole has been shown to exhibit strong anti-tumor effects in different cancer entities including AML. In the work presented here, we investigated the effect of mebendazole on expression and activity of GLI transcription factors and its anti-leukemic activity.To determine the effect of mebendazole on GLI transcription factors, we treated the AML cell lines MV4-11, MOLM-13, THP-1 and OCI-AML3 with different concentrations of mebendazole and analyzed its impact on GLI1 and GLI2 protein- and mRNA levels. Furthermore, GLI reporter assays (Cignal GLI Reporter (luc) Kit, Qiagen) were performed to determine the effect of mebendazole on the GLI1 and -2 transcriptional activity. Mebendazole strongly inhibited GLI1 and GLI2 signaling activity in a dose-dependent manner. Exemplarily, treatment with 500 nM mebendazole reduced the GLI1 and -2 transcriptional activity in all cell lines tested by 54.8 % (± 9.6) after 24h and 73.2 % (± 11.6) after 48h. We could demonstrate by Western Blotting that GLI1 and -2 protein levels were clearly reduced 24h and 48h after mebendazole exposure, whereas GLI1 and -2 mRNA levels did not decrease. These data suggest that mebendazole may increase degradation of GLI proteins via the proteasome pathway. Therefore, we evaluated the influence of the 26s proteasome inhibitor bortezomib on GLI levels after mebendazole treatment. Inhibiting the 26s proteasome with 2 nM, 5 nM and 10 nM of bortezomib increased GLI signaling activity by 13.6 % (± 8.0), 84.6 % (± 39.2) and 137.1 % (± 37.9), respectively. Furthermore, 10 nM bortezomib abolished the effect of mebendazole on GLI protein levels. Taken together, mebendazole increased the proteasomal degradation of GLI1 and GLI2. These observations were extended to samples from AML patients. After mebendazole treatment for 24h or 48h all analyzed patients had reductions of GLI1 protein levels as confirmed by Western blotting (n=4), whereas GLI1 and GLI2 mRNA levels were not changed (n=7), indicating that proteasomal degradation was operational in primary blasts as well.Evaluating the anti-leukemic effects of mebendazole, we also investigated its combination with the small molecule GLI inhibitor GANT61. We treated the AML cell lines MV4-11, MOLM-13, THP-1 and OCI-AML3 with combinations of mebendazole and GANT61 and analyzed cell proliferation, apoptosis and colony formation. Mebendazole treatment alone already resulted in decreased proliferation and colony forming capacity as well as increased apoptosis rates in a dose-dependent manner. The combination of mebendazole with the GLI inhibitor GANT61 synergistically increased the anti-proliferative effects of mebendazole on all 4 AML cell lines tested. Additionally, GANT61 further increased the effect of mebendazole on colony formation significantly. Incubation with 100 nM, 200 nM and 500 nM mebendazole inhibited the proliferation of primary blasts from AML patients by 15.1 % (± 7.5), 31.6 % (± 16.8) and 66.0 % (± 17.4), respectively (n=8). Moreover, the combination with GANT61 significantly increased these anti-proliferative effects.This work indicates that mebendazole exerts profound anti-leukemic effects by decreasing GLI1 and GLI2 intracellular levels by promoting its proteasomal degradation. Combining mebendazole with GLI1 and GLI2 inhibitors such as GANT61 enhances this effect considerably. These observations may lead to the introduction of novel treatment strategies in AML. DisclosuresStamm:Amgen Research (Munich) GmbH / Amgen Inc.: Patents & Royalties; Astellas GmbH: Other: Travel Grant. Wellbrock:Amgen Research (Munich) GmbH: Patents & Royalties. Fiedler:GSO: Other: support for meeting attendance; Gilead: Other: support for meeting attendance; Amgen: Other: support for meetíng attendance; Pfizer: Research Funding; Amgen: Research Funding; Amgen: Patents & Royalties; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; ARIAD/Incyte: Membership on an entity's Board of Directors or advisory committees, support for meeting attendance; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Other: support for meeting attendance; JAZZ Pharmaceuticals: Other: support for meeting attendance; Teva: Other: support for meeting attendance.

Effect of Ropeginterferon Alfa-2b in Prefibrotic Primary Myelofibrosis

Primary myelofibrosis (PMF) is a clonal stem cell disorder currently classified as myeloproliferative neoplasm (MPN). Median survival in PMF is estimated with 6 years, but can range from few months to several decades in early pre-fibrotic PMF. The disease course is almost always complicated by progressive anemia, symptomatic splenomegaly and severe constitutional symptoms. Causes of death include leukemic transformation with marrow failure and complications from bleeding, thrombosis, portal hypertension or infections. While PMF patients in advanced stages benefit from a JAK2 inhibitor therapy, there is no evidence for prevention of disease progression to overt MF by an early therapeutic intervention. Interferon alpha has been shown to reduce white cell counts, elevated platelet counts and improve splenomegaly and there are some observations of its disease modifying capability in the earlier phase of PMF.The aim of the present phase II study was to test the capability of a novel pegylated interferon compound (Ropeginterferon-alfa-2b) to improve hematologic parameters, splenomegaly and to monitor the course of the disease concerning clinical symptoms in a homogeneous cohort of early pre-fibrotic PMF patients. The endpoints of the study were hematological response, maintenance or improvement of quality of life and the symptom scales during a period of 2 years interferon treatment, and interferon tolerability.Twenty-five patients were included in the study. Nine patients were pretreated with pegylated interferon alfa 2b (PegIntron or Pegasys) during at least 3 years and 16 patients were interferon naïve. At start of therapy the median red blood cell count was 4.3 T/l (3.3 - 5.2); median hemoglobin level was 12.5 g/dl (9.6 - 14.7 g/dl), median WBC count was 7.7 G/l (2.44 - 27.55 G/l) and the median platelet count was 435 G/l (124 - 1161 G/l). Median LDH-level was 263.5 U/l (163 - 538 U/l) and the median spleen size was 11.2 cm (8 - 22 cm). 13 patients were JAK2 positive, and 12 patients were CALR positive. Detailed patient characteristics in 6-month intervals for pretreated and interferon naïve patients are given in Table 1.At study entry, 12 patients presented with anemia, 12 with thrombocytosis, 9 with leukocytosis, 14 with elevated LDH levels and 7 with splenomegaly. After 24 months of treatment, 6 patients improved their anemia (N=2 pretreated, 4 treatment-naïve), 6 their thrombocytosis (N=6 treatment-naïve), 7 their leukocytosis (N=7 treatment-naïve), 6 normalized LDH levels (N=1 pretreated, 5 treatment-naïve) but none showed response in the form of significant spleen size reduction. The fact that no patient showed disease progression in any single parameter after 2 years of treatment can already be considered a success.The most common side effects observed during this study include flu-like symptoms (N=7 pretreated, 15 treatment-naïve), lack of concentration (N=3 pretreated, 8 treatment-naïve), pruritus (N=2 pretreated, 7 treatment-naïve), infections (N=2 pretreated, 5 treatment-naïve) and bleeding (N=2 pretreated, 4 treatment-naïve). Generally, pretreated patients suffered from fewer side effects than treatment-naïve patients in every category. This may be due to the fact that those patients had already been exposed to an interferon in previous treatment regimen. Two patients had to be withdrawn from the study after approximately 12 months due to psychological side effects, both from the treatment-naïve group.Additionally to conventional symptom screening, the EORTC-QLQ-C30 questionnaire was used to assess therapy-response. With this tool, function scales, symptom scales and quality-of-life were recorded. After 24 months of treatment, almost every category showed improvement for both pretreated and treatment-naïve patients. Detailed results about symptom scales are shown in Figure 1.In conclusion, the data demonstrate that Ropeginterferon-alfa-2b is able not only to induce hematologic responses but also to improve constitutional symptoms and overall quality of life in prefibrotic PMF. Thus ropeginterferon-alfa-2b warrant further investigation as a treatment option in prefibrotic PMF, where only very limited alternatives exist. [Display omitted] DisclosuresGisslinger:Shire: Consultancy, Honoraria; Janssen Cilag: Consultancy, Honoraria; Novartis: Honoraria, Research Funding; AOP Orphan Pharmaceuticals AG: Consultancy, Honoraria, Research Funding. Krejcy:AOP Orphan Pharmaceuticals: Employment. Widmann:AOP Orphan Pharmaceuticals: Employment. Kralovics:MyeloPro Diagnostics and Research GmbH: Equity Ownership. Krauth:Novartis: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Celgene: Consultancy, Honoraria.