Kurzfassung: Abstract 771 Introduction. Platelets are activated early during the reperfusion of ischemic myocardium, potentially exacerbating the extent of ischemia/reperfusion (I/R) injury. We have recently shown (Zhu, et al., PNAS 2007) that platelets express the semaphorin family member, sema4D, as do T-cells. Sema4D is a cell surface protein whose receptors are expressed by B-cells, monocytes and endothelial cells as well as platelets. Loss of sema4D expression in mice causes a defect in signaling downstream of the platelet collagen receptor, glycoprotein (GP) VI, inhibiting platelet function in vitro and in vivo, and reducing the extent of platelet hyperresponsiveness and atherothrombosis in the setting of dyslipidemia (Zhu, et al. ATVB 2009). Because of the role played by platelets, leukocytes and endothelial cells in reperfusion injury, here we asked whether loss of sema4D expression can also protect the heart, reducing the extent of damage following temporary ischemia. Methods. The left anterior descending coronary artery of anaesthetized mice was ligated for 45 min. After reperfusion for 48 hours, the mice were re-anesthetized and perfused with 2,3,5-triphenyltetrazolium chloride to measure the area of infarction. Fluorescent microspheres were used to delineate the area at risk. Comparisons were made between sema4D(−/−) and wild type mice produced by breeding sema4D(+/−) heterozygotes. Results. Although there was no difference between the sema4D(−/−) and WT mice with respect to either heart size or area at risk, we observed a substantial (57%) decrease in infarct size in the sema4D(−/−) mice expressed as a fraction of the area at risk (N=7-9, p 〈 0.005). Since sema4D is shed from the surface of activated platelets and T-cells by the metalloprotease, ADAM17, producing a large bioactive fragment, we next asked whether the protection against ischemia/reperfusion injury conferred by the sema4D knockout is due to the loss of cell-associated or soluble sema4D. Chimeric mice were produced in which hematopoiesis was reconstituted in irradiated sema4D(+/+) mice using fetal liver cells from mouse embryos that lack functional ADAM17. This produces mice in which sema4D is expressed as usual in the hematopoietic lineages, but unable to be shed. Chimerism, inhibition of sema4D shedding and recovery of normal cell counts were confirmed after transplantation. The ischemia/reperfusion studies were repeated comparing chimeras reconstituted with ADAM17-deficient and ADAM17-replete fetal liver cells. In contrast to the sema4D knockout, the extent of infarction was the same whether or not ADAM17 was functional and sema4D was shed. Conclusions. Although the role of sema4D and its receptors have been studied most extensively in the context of T-cell interactions with B-cells, our previous studies have made a case for the involvement of sema4D in platelet:platelet and platelet:endothelial cell interactions. We now show for the first time that 1) loss of sema4D expression in mice confers protection against ischemia/reperfusion injury in the myocardium, and 2) preventing the formation of soluble, bioactive sema4D is insufficient to recapitulate this effect. Since sema4D and its receptors are expressed on more than just platelets, it cannot be concluded that the observed protection in the knockout is solely due to the absence of platelet sema4D. However, experience with other knockouts that reduce platelet function suggests that the defects that we have observed in sema4D(−/−) platelet function are likely to contribute. Regardless of whether expression on platelets is entirely or only partly responsible for the observed phenotype, sema4D is an interesting target for therapeutic intervention. Disclosures: No relevant conflicts of interest to declare.

Kurzfassung: There has been controversy over the existence of rare somatic stem cells in mouse and human bone marrow and in human umbilical cord blood that have been described to be pluripotent. These cells have been observed to lack expression of CD45 and blood cell lineage markers (Lin-), to express markers associated with both hematopoietic (CD34, CD133) and pluripotent (Oct4, Nanog) stem cells, to be smaller than a blood lymphocyte, and have been associated with the ability to differentiate into cells and tissues of all three germ layers. They have been given a variety of names, including Very Small Embryonic/Epiblast-Like (VSEL) cells. Methods and Results To better characterize these cells, we fractionated G-CSF mobilized adult peripheral blood by elutriation, CD34/CD133 immunomagnetic selection (Miltenyi Biotec, Inc., STEMCELL Technologies, Inc.) and fluorescence-activated cell sorting (FACS) using a MoFlo XDP cell sorter (Beckman Coulter, Inc.). Cell fractions were analyzed on a Beckman Coulter Gallios flow cytometer using a 6-color cocktail of antibodies to CD45, CD34, CD133 and blood cell lineage markers, together with membrane-permeable (DRAQ5) and impermeable (7-AAD) nuclear dyes that distinguish live nucleated cells from dead cells, extracellular vesicles and cell debris. We observe that over 95% of Lin- CD45- flow cytometry events are extracellular vesicles rather than nucleated cells, and have isolated a population of Lin- CD45- CD34+ vesicles from the earliest elutriation fractions ( 〈 35 ml/min counterflow). Rare Lin- CD45- live nucleated cells are also clearly evident, many of which express CD34 but not CD133. This population of Lin- CD45- CD34+ CD133- cells makes up approximately 0.003% of the mononuclear cell population in G-CSF mobilized peripheral blood (1 in 300,000 mononuclear cells, or approximately 1 for every 1,000 CD34+ CD45+ hematopoietic stem/progenitor cells). Lin- CD45- CD133+ live nucleated cells are also identified but are considerably more rare (approximately 1 in 10 million mononuclear cells). Similar cell and vesicle populations are also found in umbilical cord blood, although with frequencies about 10-fold higher than in mobilized adult blood. To begin to characterize these cell and vesicle fractions, we isolated total RNA from FACS-sorted Lin- CD45- CD34/CD133+ (CD34+ and/or CD133+) cells for miRNA expression profiling (Agilent SurePrint G3 Human v16 microRNA 8x60K microarray, representing 1205 Human miRNAs, 1199 verified as real miRNAs in miRbase 18). Array data were processed using a proprietary normalization algorithm (Sistemic, Ltd.) to generate miRNA expression profiles which were analyzed by microRNA-based fingerprinting (SistemQC™). A robust miRNA profile was generated from the initial Lin- CD45- CD34/CD133+ cell sample, with 107 miRNAs reliably detected (a number consistent with other cellular profiles). The detected miRNAs showed a range of expression levels and were expressed well above the limit of detection for the arrays. Further samples will be analyzed to confirm these preliminary findings. Conclusion The Lin- CD45- population observed by flow cytometry analysis of human mobilized peripheral blood and umbilical cord blood is heterogeneous, and made up of distinct populations of vesicles and live nucleated cells that variably express CD34 and CD133. The ability to determine miRNA profiles from rare sorted populations such as Lin- CD45- CD34/CD133+ cells will enable the possible further understanding of the function of these cells, as well as the role of miRNAs in regulating their cellular processes. It is also hoped that the data will enhance the understanding of the potential clinical utility of such cells isolated from human blood products. Disclosures: Jiang: NeoStem, Inc.: Employment. Mallinson:Sistemic, Ltd.: Employment, Equity Ownership. Olijnyk:Sistemic, Ltd.: Employment. Paterson:Sistemic, Ltd.: Employment. Ridha:Sistemic, Ltd.: Employment. Tang:NeoStem, Inc.: Employment. O'Brien:Sistemic, Ltd.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Fong:NeoStem, Inc.: Employment. O'Neill:NeoStem, Inc.: Employment.

Kurzfassung: Although much is known about extrinsic regulators of platelet function such as nitric oxide and prostaglandin I2 (PGI2), considerably less is known about intrinsic mechanisms that prevent overly robust platelet activation after vascular injury. Here we provide the first evidence that regulators of G-protein signaling (RGS) proteins serve this role in platelets, using mice with a G184S substitution in Gi2α that blocks RGS/Gi2 interactions to examine the consequences of lifting constraints on Gi2-dependent signaling without altering receptor:effector coupling. The results show that the Gi2α(G184S) allele enhances platelet aggregation in vitro and increases platelet accumulation after vascular injury when expressed either as a global knock-in or limited to hematopoietic cells. Biochemical studies show that these changes occur in concert with an attenuated rise in cyclic adenosine monophosphate levels in response to prostacyclin and a substantial increase in basal Akt activation. In contrast, basal cyclic adenosine monophosphate (cAMP) levels, agonist-stimulated increases in [Ca++]i, Rap1 activation, and α-granule secretion were unaffected. Collectively, these observations (1) demonstrate an active role for RGS proteins in regulating platelet responsiveness, (2) show that this occurs in a pathway-selective manner, and (3) suggest that RGS proteins help to prevent unwarranted platelet activation as well as limiting the magnitude of the normal hemostatic response.

Kurzfassung: Hydroxychloroquine (HCQ) is an immunosuppressive lysosomotropic amine that has activity against graft-versus-host disease (GVHD). We previously reported low incidences of acute GVHD in unrelated donor transplant recipients who received prophylactic HCQ in addition to standard GVHD prophylaxis (BBMT2003; 9: 714–721). We herein report results of a single-institution phase III trial, in which 95 recipients of matched sibling allogeneic peripheral blood stem cell transplantation were randomized to receive, in a double-blind fashion, and in addition to prophylactic cyclosporine A (CSA), HCQ or placebo starting 21 days pre-transplant and continued until d+365. HCQ was very well tolerated and not associated with side effects. The addition of HCQ had no effects on lymphocyte subsets both pre- and post-transplant. Overall, the incidence of acute GVHD was 59% in both arms, and severe acute GVHD occurred in 11% (HCQ) and 14% (placebo) (p=0.76). Sixty-one and 46% of patients developed chronic GVHD in the placebo and the HCQ arms, respectively (p = 0.15). With a median follow-up of 18 months, relapse-free and overall survivals were comparable in both groups. In summary, in this randomized trial, the addition of HCQ to single agent CSA was not associated with a reduction of either acute or chronic GVHD; additionally, no significant effects on relapses or survival were observed.

Kurzfassung: Introduction Therapeutic products with blood coagulation factor VIII (FVIII) have a wide range of protein contents per activity unit (or IU/mg, specific activity), available from previous studies (Lin et al, 2004; Butenas et al, 2009) and the prescribing information. The wide range of the specific activities seems to be more pronounced in recombinant FVIII products (rFVIII) compared to plasma-derived FVIII, implying presence of protein with altered structure and biochemical properties. In particular, rFVIII products were reported to contain a protein fraction (FVIII*), unable to bind von Willebrand factor (VWF) without functional activity (Lin et al, 2004;Ofosu et al 2012). Furthermore, FVIII* may represent a risk factor for development of FVIII inhibitors in Hemophilia A patients, as the binding to VWF was shown to reduce FVIII immunogenicity in a tissue culture and mice model systems (Gangadharan et al, 2017; Muczynski et al, 2018). Due to these reasons, FVIII* can be defined as an impurity, which needs to be understood and controlled in rFVIII products. Study objective To develop a methodology to isolate FVIII* from rFVIII samples and to characterize this fraction in various rFVIII products. Experimental design Using immobilized VWF affinity chromatography (IVAC) for analysis of FVIII samples, protein fractions collected from (i) the column flow-through (FVIIIFT, corresponding to FVIII*) and (ii) the column-bound and eluted fraction (FVIIIEL) were characterized using polyacrylamide gel electrophoresis (PAGE) gels followed by silver-staining and immunoblotting, FVIII activity test, surface plasmon resonance, mass spectrometry, and for plasma clearance in mice. Results A robust IVAC methodology was developed. Using this method, we isolated the FVIIIFT and FVIIIEL from all ten third-generation rFVIII products marketed in the USA by study time. These products represent all current pharmaceutical variants of rFVIII including full-size FVIII, B-domain deleted (truncated) FVIII, Fc-fused FVIII, single-chained FVIII, and PEGylated protein, including those with the extended plasma half-life. FVIIIFT was found in all rFVIII products at levels up to 22% of total protein. Compared to FVIIIEL, FVIIIFT had similar pattern of polypeptide bands by PAGE, but lower functional activity, significantly reduced sulfation at Tyr1680 important for VWF binding, moderately decreased interaction with recombinant cluster II of a low-density lipoprotein receptor related protein 1 (a major clearance receptor of FVIII), and approximately 3-times faster clearance in mice (Figure 1). Conclusions Our results show that the FVIII* structure is generally similar to that of the major fraction of rFVIII, while it differs by microheterogeneity in post-translational modifications and possibly local misfolding. The data suggest that upon administration of a rFVIII product in patients, its FVIII* fraction is rapidly removed from the circulation, resulting in a decrease of the effective dosage. Our findings demonstrate a potential of IVAC to control FVIII* fraction in rFVIII products, including its removal from the products during their manufacture. These applications may lead to achieving better quality and efficacy of rFVIII products including reduction of their immunogenicity for improving the care of Hemophilia A. Disclosures No relevant conflicts of interest to declare.

Kurzfassung: Systemic inflammation can lead to coagulopathy and disseminated intravascular coagulation (DIC). In prior studies, the recombinant A2 domain of human von Willebrand factor (VWF; A2 protein) attenuated DIC and decreased mortality in lipopolysaccharide (LPS)-treated mice. Here, we performed studies to dissect the mechanism by which the A2 protein moderates DIC. We used confocal microscopy to analyze the fibrin clot structure in plasma from healthy humans and endotoxemic mice, turbidity assays to examine fibrin polymerization, and a murine model for LPS-induced DIC and introduced a loss-of-function mutation into the A2 protein for fibrin. The mutation of the residue E1567 located in the α2 helix of the folded A2 domain of VWF inhibited binding activity for fibrin, possibly mapping a novel region containing a putative binding site for fibrin. The A2 protein increased the initial rate of change of fibrin polymerization, intercalated into the fibrin network, and modified the resultant clot structure in vitro. Furthermore, ex vivo experiments using plasma from mice with endotoxemia treated with the A2 protein revealed an increased rate of fibrin formation and an altered clot structure as compared with plasma from nontreated sick animals. Moreover, and in contrast to the A2 mutant, the A2 protein improved survival and reduced fibrin deposition and microvascular thrombosis in mice with endotoxemia-induced DIC. Importantly, in vivo and in vitro studies indicated that the A2 protein did not affect experimental thrombosis. Thus, we provide evidence for a novel treatment to attenuate systemic inflammation-induced coagulopathy/DIC via targeting fibrin formation, without an increased risk for bleeding.

Kurzfassung: We recently reported that Semaphorin 4D (Sema4D) and its receptors are expressed on the platelet surface and showed that Sema4D(−/−) mice have a selective defect in collagen-induced platelet aggregation and an impaired vascular injury response. Here we investigated the mechanisms involved, tested the role of platelet-platelet contacts in Sema4D-mediated events, and examined the relationship between Sema4D-dependent signaling and integrin αIIbβ3 outside-in signaling. The results show that spleen tyrosine kinase (Syk) activation, an early step in collagen signaling via the glycoprotein VI (GPVI)/FcRγ complex, is greatly reduced in Sema4D(−/−) platelets and can be restored by adding soluble Sema4D. Earlier events, including FcRγ phosphorylation, occur normally; later events are impaired. In contrast, when engagement of αIIbβ3 was blocked, Sema4D(−/−) and control platelets were indistinguishable in assays of Syk activation, adhesion, spreading on collagen, and activation of αIIbβ3. Finally, we found that, unlike the Sema4D knockout, αIIbβ3 blockade inhibited FcRγ phosphorylation and that stimulating aggregation with Mn2+ failed to normalize Syk activation in the absence of Sema4D. Collectively, these results show that αIIbβ3 and Sema4D jointly promote collagen responses by amplifying Syk activation, partly by forming integrin-mediated contacts that enable the binding of Sema4D to its receptors and partly through integrin outside-in signaling. These 2 processes are interdependent, but distinguishable.

Kurzfassung: AMR-001, an autologous CD34+ cell product derived from mini-marrow harvest, is currently undergoing Phase II trials to treat acute myocardial infarction (AMI). AMR-001 is administered to the patient by infusion via the infarct related artery within five to ten days following coronary artery stenting post AMI. At the time of infusion, it is believed that the infarct-region SDF-1 (stromal derived factor) levels are peaked and scar formation has not yet occurred. It was found that, in addition to the quantity of CD34+ cells infused, improvement in cardiac perfusion and infarct size correlated with the mobility potential of CD34+ cells mediated by a SDF-1 gradient (Quyyumi et al, Am Heart J 2011, 161:98–105). We have developed a cell based in vitro mobility assay as a potential potency release assay for AMR-001. However, this assay is not suitable for a Phase III or commercial scale release assay due to the length of the assay, high skill level required to perform, and variability. To develop a more robust assay, we have initiated a study to identify potential microRNAs (miRNAs) that may be used as biomarkers for CD34+ cell SDF-1 driven migration. Our preliminary results suggest CD34+ cells with different mobility potentials may be characterized by miRNA fingerprinting. Methods Cryopreserved purified CD34+ cells derived from bone marrow of healthy donors were purchased from a commercial vendor. Thawed CD34+ cells were washed and the cells were assayed in an in vitro transwell system (Jo et al, J Clin Invest 2000, 105:101-111). The trans-membrane migration of CD34+ cells into the lower chamber in the presence of SDF-1, as well as the non-mobilized CD34+ cells in the upper chamber, were collected after 4 hours incubation at 37°C. Total RNA of the cells was isolated and the miRNA expression profile was analyzed using SurePrint G3 Human v16 microRNA 8x60K microarray slide (Agilent, Santa Clara, CA). A normalization algorithm was used to generate miRNA expression profiles (SistemQC™, Sistemic, Ltd) for the characterization of untreated cells, the mobilized population that migrate towards SDF-1, and non-mobilized population; from two independent donors. Results Two hundred and four (204) miRNAs were reliably detected across the cell samples. The mobilized cells had different miRNA profiles compared with non-mobilized/untreated cells. Hierarchical cluster analysis showed that mobilized cells grouped separately from the non-mobilized/untreated cells. Conclusion Analysis of the miRNA profiles of the CD34+ cells across two independent donors, identified a number of key miRNAs (kmiRs™) that represent possible markers for a mobility phenotype. Additional samples will be analyzed to confirm these preliminary findings. This approach will enable the identification of markers associated with mobility potential of CD34+ cells and the potential development of a molecular biomarker assay for potency. Disclosures: Warbington: Progenitor Cell Therapy, LLC: Employment. Weinstein:Progenitor Cell Therapy, LLC: Employment. Mallinson:Sistemic, Ltd.: Employment, Equity Ownership. Olijnyk:Sistemic, Ltd.: Employment. Paterson:Sistemic, Ltd.: Employment. Ridha:Sistemic, Ltd.: Employment. O'Brien:Sistemic, Ltd.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Lin:Progenitor Cell Therapy, LLC: Employment. LeBlon:Progenitor Cell Therapy, LLC: Employment. Fong:NeoStem, Inc.: Employment. Chan:Progenitor Cell Therapy, LLC: Employment.

Kurzfassung: Background: Patients with chronic lymphocytic leukemia (CLL) with deletion of the short arm of chromosome 17 (del 17p) follow an aggressive clinical course and demonstrate a median survival of less than 2 years in the relapsed/refractory (R/R) setting. Ibrutinib (ImbruvicaTM), a first-in-class Bruton's tyrosine kinase (BTK) inhibitor, has been approved for previously treated patients with CLL and for patients with del 17p CLL. We report results from the primary analysis of the Phase II RESONATETM-17 (PCYC-1117-CA) study, designed to evaluate the efficacy and safety of single-agent ibrutinib for treatment of patients with R/R del 17p CLL or small lymphocytic leukemia (SLL). Methods: Patients with del 17p CLL or SLL who failed at least one therapy were enrolled to receive 420 mg oral ibrutinib once daily until progression. All patients receiving at least one dose of ibrutinib were included in the analysis. The primary endpoint was overall response rate (ORR) per an independent review committee (IRC). Other endpoints included duration of response (DOR), progression-free survival (PFS), and safety of ibrutinib. Results: Among 144 treated patients (137 with CLL, 7 with SLL), the median age was 64 (48% 65 years or older) and all had del 17p. Baseline characteristics included 63% of patients with Rai Stage III or IV disease, 49% with bulky lymphadenopathy of at least 5 cm, and 10% with lymphadenopathy of least 10 cm. The median baseline absolute lymphocyte count (ALC) was 32.9 x 109/L with 57% of patients with a baseline ALC at least 25.0 x 109/L. Baseline beta-2 microglobulin levels were at least 3.5 mg/L in 78% of patients (range 1.8-19.8 mg/L), and lactate dehydrogenase levels were at least 350 U/L in 24% of patients (range 127-1979 U/L). A median of 2 prior therapies (range 1-7) was reported. Investigator-assessed ORR was 82.6% including 17.4% partial response with lymphocytosis (PR-L). Complete response (CR)/complete response with incomplete bone marrow recovery (CRi) were reported in 3 patients. IRC-assessed ORR is pending. At a median follow up of 13.0 months (range 0.5-16.7 months), the median PFS (Figure 1) and DOR by investigator determination had not been reached. At 12 months, 79.3% were alive and progression-free, and 88.3% of responders were progression-free. Progressive disease was reported in 20 patients (13.9%). Richter transformation was reported in 11 of these patients (7.6%), 7 of the cases occurring within the first 24 weeks of treatment. Prolymphocytic leukemia was reported in 1 patient. The most frequently reported adverse events (AE) of any grade were diarrhea (36%; 2% Grade 3-4), fatigue (30%; 1% Grade 3-4), cough (24%; 1% Grade 3-4), and arthralgia (22%; 1% Grade 3-4). Atrial fibrillation of any grade was reported in 11 patients (7.6%; 3.5% Grade 3-4). Seven patients reported basal or squamous cell skin cancer and 1 patient had plasma cell myeloma. Most frequently reported Grade 3-4 AEs were neutropenia (14%), anemia (8%), pneumonia (8%), and hypertension (8%). Major hemorrhage was reported in 7 patients (4.9%, all Grade 2 or 3). Study treatment was discontinued in 16 patients (11.1%) due to AEs with 8 eventually having fatal events (pneumonia, sepsis, myocardial or renal infarction, health deterioration). At the time of data cut, the median treatment duration was 11.1 months, and 101 of 144 patients (70%) continued treatment with ibrutinib. Conclusions: In the largest prospective trial dedicated to the study of del 17p CLL/SLL, ibrutinib demonstrated marked efficacy in terms of ORR, DOR, and PFS, with a favorable risk-benefit profile. At a median follow up of 13 months, the median DOR had not yet been reached; 79.3% of patients remained progression-free at 12 months, consistent with efficacy observed in earlier studies (Byrd, NEJM 2013;369:32-42). The PFS in this previously treated population compares favorably to that of treatment-naïve del 17p CLL patients receiving fludarabine, cyclophosphamide, and rituximab (FCR) (Hallek, Lancet 2010;376:1164-74) or alemtuzumab (Hillmen, J Clin Oncol 2007;10:5616-23) with median PFS of 11 months. The AEs are consistent with those previously reported for ibrutinib (Byrd, NEJM 2014;371:213-23). These results support ibrutinib as an effective therapy for patients with del 17p CLL/SLL. Figure 1 Figure 1. Disclosures O'Brien: Amgen, Celgene, GSK: Consultancy; CLL Global Research Foundation: Membership on an entity's Board of Directors or advisory committees; Emergent, Genentech, Gilead, Infinity, Pharmacyclics, Spectrum: Consultancy, Research Funding; MorphoSys, Acerta, TG Therapeutics: Research Funding. Jones:Pharmacyclics: Consultancy, Research Funding. Coutre:Janssen, Pharmacyclics: Honoraria, Research Funding. Mato:Pharamcyclics, Genentech, Celegene, Millennium : Speakers Bureau. Hillmen:Pharmacyclics, Janssen, Gilead, Roche: Honoraria, Research Funding. Tam:Pharmacyclics and Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Siddiqi:Janssen: Speakers Bureau. Furman:Pharmacyclics: Consultancy, Speakers Bureau. Brown:Sanofi, Onyx, Vertex, Novartis, Boehringer, GSK, Roche/Genentech, Emergent, Morphosys, Celgene, Janssen, Pharmacyclics, Gilead: Consultancy. Stevens-Brogan:Pharmacyclics: Employment. Li:Pharmacyclics: Employment. Fardis:Pharmacyclics: Employment. Clow:Pharmacyclics: Employment. James:Pharmacyclics: Employment. Chu:Pharmacyclics: Employment, Equity Ownership. Hallek:Janssen, Pharmacyclics: Consultancy, Research Funding. Stilgenbauer:Pharmacyclics, Janssen Cilag: Consultancy, Honoraria, Research Funding.