Background Coagulation factor XI ( FXI ) contributes to the development of thrombosis but appears to play only a minor role in hemostasis and is therefore an attractive anticoagulant drug target. Objectives To evaluate the safety, pharmacodynamic, and pharmacokinetic properties of BAY 1213790, a fully human immunoglobulin (Ig) G1 antibody targeting activated coagulation FXI ( FXI a), in healthy men. Methods In this phase 1, single‐blind, parallel‐group, placebo‐controlled, dose‐escalation study, 83 healthy Caucasian men were randomized 4:1 to receive a single 60‐minute intravenous infusion of BAY 1213790 (0.015‐10 mg/kg) or placebo. Adverse events, pharmacodynamic parameters (including activated partial thromboplastin time [ aPTT ]) and pharmacokinetic parameters were determined. Volunteers were followed up for 150 days. Results BAY 1213790 demonstrated favorable safety and tolerability; there were no observed cases of bleeding or clinically relevant antidrug antibody formation. One volunteer (1.2%) experienced an infusion reaction. Following intravenous administration of BAY 1213790, dose‐dependent increases in aPTT (maximal mean increase relative to baseline: 1.85 [conventional method] and 2.17 [kaolin‐triggered method]) and rotational thromboelastometry whole blood clotting time were observed, as well as dose‐dependent reductions in FXI activity. Bleeding times did not increase following administration of BAY 1213790 and were similar for all dose cohorts, including placebo. Measurable and dose‐dependent increases in systemic exposure were detected for all doses of BAY 1213790 of 0.06 mg/kg or higher. Conclusions Based on these safety, pharmacodynamic, and pharmacokinetic results, further evaluation of BAY 1213790 in patients with, or at risk of, thrombosis is warranted.

Introduction This X-VeRT (eXplore the efficacy and safety of once-daily oral riVaroxaban for the prevention of caRdiovascular events in patients with nonvalvular aTrial fibrillation scheduled for cardioversion) substudy evaluated the effects of treatment with rivaroxaban or a vitamin-K antagonist (VKA) on levels of biomarkers of coagulation (D-dimer, thrombin–antithrombin III complex [TAT] and prothrombin fragment [F1.2]) and inflammation (high sensitivity C-reactive protein [hs-CRP] and high-sensitivity interleukin-6 [hs-IL-6]) in patients with atrial fibrillation (AF) who were scheduled for cardioversion and had not received adequate anticoagulation at baseline (defined as, in the 21 days before randomization: no oral anticoagulant; international normalized ratio <2.0 with VKA treatment; or <80% compliance with non-VKA oral anticoagulant treatment). Methods Samples for biomarker analysis were taken at baseline (n = 958) and treatment completion (42 days after cardioversion; n = 918). The influence of clinical characteristics on baseline biomarker levels and the effect of treatment on changes in biomarker levels were evaluated using linear and logistic models. Results Baseline levels of some biomarkers were significantly associated with type of AF (D-dimer and hs-IL-6) and with history of congestive heart failure (hs-CRP, D-dimer, and hs-IL-6). Rivaroxaban and VKA treatments were associated with reductions from baseline in levels of D-dimer (−32.3 and −37.6%, respectively), TAT (−28.0 and −23.1%, respectively), hs-CRP (−12.5 and −17.9%, respectively), and hs-IL-6 (−9.2 and −9.8%, respectively). F1.2 levels were reduced from baseline in patients receiving a VKA (−53.0%) but not in those receiving rivaroxaban (2.7%). Conclusion Anticoagulation with rivaroxaban reduced levels of key inflammation and coagulation biomarkers to a similar extent as VKAs, with the exception of F1.2. Further investigation to confirm the value of these biomarkers in patients with AF is merited.

Tumors develop when infiltrating immune cells contribute growth stimuli, and cancer cells are selected to survive within such a cytotoxic microenvironment. One possible immune-escape mechanism is the upregulation of PI-9 (Serpin B9) within cancer cells. This serine proteinase inhibitor selectively inactivates apoptosis-inducing granzyme B (GrB) from cytotoxic granules of innate immune cells. We demonstrate that most classical Hodgkin lymphoma (cHL)-derived cell lines express PI-9, which protects them against the GrB attack and thereby renders them resistant against GrB-based immunotherapeutics. To circumvent this disadvantage, we developed PI-9-insensitive human GrB mutants as fusion proteins to target the Hodgkin-selective receptor CD30. In contrast to the wild-type GrB, a R201K point-mutated GrB construct most efficiently killed PI-9-positive and -negative cHL cells. This was tested in vitro and also in vivo whereby a novel optical imaging-based tumor model with HL cell line L428 was applied. Therefore, this variant, as part of the next generation immunotherapeutics, also named cytolytic fusion proteins showing reduced immunogenicity, is a promising molecule for (targeted) therapy of patients with relapsing malignancies, such as cHL, and possibly other PI-9-positive malignancies, such as breast or lung carcinoma.

Human granzyme B (hGB) is a serine protease involved in immune-mediated apoptosis. Its cytotoxicity makes it potentially applicable in cancer therapy. However, the effectiveness of hGB can be hampered by the cytosolic expression of a natural protein inhibitor, human Serpin B9 (hSB9). Here, we used computational approaches to identify hGB mutations that can affect its binding to hSB9 without significantly decreasing its catalytic efficiency. Alanine-scanning calculations allowed us to identify residues of hGB important for the interaction with hSB9. Some variants were selected, and molecular dynamic simulations on the mutated hGB in complex with hSB9 in aqueous solution were carried out to investigate the effect of these variants on the stability of the complex. The R28K, R201A, and R201K mutants significantly destabilized the interaction of the protein with hSB9. Consistently, all of these variants also retained their activity in the presence of the Serpin B9 inhibitor in subsequent in vitro assays of wild-type and mutated hGB. In particular, the activity of R201K hGB with and without Serpin B9 is very similar to that of the wild-type protein. Hence, R201K hGB emerges as a promising species for antitumoral therapy applications.

CMML (chronic myelomonocytic leukemia) belongs to the group of myeloid neoplasms known as myelodysplastic and myeloproliferative diseases. In some patients with a history of CMML, the disease transforms to acute myelomonocytic leukemia (AMML). There are no specific treatment options for patients suffering from CMML except for supportive care and DNA methyltransferase inhibitors in patients with advanced disease. New treatment strategies are urgently required, so we have investigated the use of immunotherapeutic directed cytolytic fusion proteins (CFPs), which are chimeric proteins comprising a selective domain and a toxic component (preferably of human origin to avoid immunogenicity). The human serine protease granzyme B is a prominent candidate for tumor immunotherapy because it is expressed in cytotoxic T lymphocytes and natural killer cells. Here, we report the use of CD64 as a novel target for specific CMML and AMML therapy, and correlate CD64 expression with typical surface markers representing these diseases. We demonstrate that CD64-specific human CFPs kill CMML and AMML cells ex vivo, and that the mutant granzyme B protein R201K is more cytotoxic than the wild-type enzyme in the presence of the granzyme B inhibitor PI9. Besides, the human CFP based on the granzyme B mutant was also able to kill AMML or CMML probes resistant to Pseudomonas exotoxin A.The clonal hematopoietic stem cell disorder CMML (chronic myelomonocytic leukemia), is a highly aggressive and resistant form of leukemia. It is characterized by persistent monocytosis in the peripheral blood, at least one dysplasia component in the bone marrow, and <20% promonocytes and blasts in peripheral blood and bone marrow. 1 CMML can be classified further according to blast numbers, that is, CMML1 (<5% blasts in peripheral blood and <10% blasts in bone marrow) and CMML2 (<20% blasts in peripheral blood and 10-19% blasts in bone marrow).2,3 The disease occurs mainly in elderly people with a median survival of 15-20 months and leukemic transformation rates of 15-30%, both factors depending on the disease subtype. 4 Other diagnostic criteria include immunophenotyping based on the overlapping antigen expression patterns in CMML and acute myelomonocytic leukemia (AMML). The expression of CD56 combined with reductions in the levels of myeloid markers such as CD15 and CD13 is a unique signature of CMML monocytes.5 Furthermore, CD14 is expressed at higher levels on bone marrow monocytes in CMML compared with reactive monocytosis and normal marrow samples. 5 High levels of CD33 have also been reported. 6 Clonal cytogenetic abnormalities are found in 20-40% of CMML patients, but these do not appear to be specific. [7][8][9] The close relationship between CMML and other myelodysplastic (MDS)/MPNs makes correct diagnosis and treatment a significant challenge. Novel molecular markers described more recently include specific alleles of TET2 and CBL, but these

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Heterogeneous patient populations, complex pharmacology and low recruitment rates in the Intensive Care Unit (ICU) have led to the failure of many clinical trials. Recently, machine learning (ML) emerged as a new technology to process and identify big data relationships, enabling a new era in clinical trial design. In this study, we designed a ML model for predictively stratifying acute respiratory distress syndrome (ARDS) patients, ultimately reducing the required number of patients by increasing statistical power through cohort homogeneity. From the Philips eICU Research Institute (eRI) database, no less than 51,555 ARDS patients were extracted. We defined three subpopulations by outcome: (1) rapid death, (2) spontaneous recovery, and (3) long-stay patients. A retrospective univariate analysis identified highly predictive variables for each outcome. All 220 variables were used to determine the most accurate and generalizable model to predict long-stay patients. Multiclass gradient boosting was identified as the best-performing ML model. Whereas alterations in pH, bicarbonate or lactate proved to be strong predictors for rapid death in the univariate analysis, only the multivariate ML model was able to reliably differentiate the disease course of the long-stay outcome population (AUC of 0.77). We demonstrate the feasibility of prospective patient stratification using ML algorithms in the by far largest ARDS cohort reported to date. Our algorithm can identify patients with sufficiently long ARDS episodes to allow time for patients to respond to therapy, increasing statistical power. Further, early enrollment alerts may increase recruitment rate.

Human cytolytic fusion proteins (hCFPs) are comprised of a specific cell-surface-binding moiety and an effector molecule of human origin. In contrast to common immunotoxins, including bacterial or plant toxins, they are considered not to be immunogenic. Two examples for human pro-apoptotic effector proteins are the serine protease Granzyme B and the RNase Angiogenin. Pre-clinical testing of functionality in in vitro and in vivo studies is essential for therapeutics. Establishing relevant animal models that have predictive value for therapeutic success is a great challenge in biomedical research. In this study, we investigated the species-dependent cytotoxic activity of two hCFPs prior to their application in a murine inflammation model. We found that in vitro and ex vivo either hCFP was able to kill human cells only, leaving murine cells unaffected. In contrast, no species-dependency was found for the bacterial Pseudomonas exotoxin A based immunotoxin H22(scFv)-ETA'. This species-dependent functioning has to be carefully considered when performing pre-clinical studies in animal models.