Sevuparin Blocks Sickle Blood Cell Adhesion and Sickle-Leukocyte Rolling on Immobilized L-Selectin in a Dose Dependent Manner

Abstract Background: The cause and continuation of vaso-occlusion in sickle cell disease (SCD) are fueled by the sickle-Red Blood Cells interactions with multiple other cell populations, promoting inflammation, obstructing the vasculature, and injuring the endothelium, leading to broad manifestations that affect most vital organs. Recent studies have identified multiple cellular components and molecular factors that contribute to the pathophysiology of SCD as reviewed by Zhang et al 2016 in Blood. It is likely that a multi-targeted approach for addressing SCD vaso-occlusion will be required to achieve the best clinical outcome. Sevuparin (DF02), a novel drug in Phase 2 for acute treatment of vaso-occlusive crisis in SCD (NCT02515838), is a polysaccharide blocking abnormal adhesion and thereby normalizing obstructed blood flow. In vitro and in vivo studies have shown potent anti-adhesive effects with a multimodal mechanism of action. In this study, we evaluate the effects of sevuparin on the adhesion of sickle whole blood from individual patients to endothelial cells (HUVECs) and vascular cell adhesion molecule-1 (VCAM-1) (Flow Firm Adhesion) and sickle-leukocyte rolling adhesion on L-selectin (Flow Dynamic Adhesion) using a standardized microfluidic flow-based adhesion assay. Methods: Blood was obtained from homozygous SCD patients (n = 12, age range 15-25yrs) in sodium citrate after obtaining informed consent. A comprehensive assessment of the effect of sevuparin on whole blood adhesive properties during simulated blood flow was assessed using standardized Flow Firm Adhesion and Flow Dynamic Adhesion assays (Functional Fluidics, Detroit MI). Flow Firm Adhesion: Whole blood firm adhesion was measured during physiologic flow in microfluidic channels (Fluxion-Bioflux 1000, San Francisco, CA) coated with either VCAM-1 or cultured HUVECs. HUVECs were activated by TNF-alpha (25ng/mL x 24 hrs.) and Histamine (100mM x 10min) prior to the assay. Whole blood was treated with increasing doses of sevuparin (0, 3, 7, 21, 200µg/mL) for 30 min. Dose response of whole blood adhesion index (cells/mm2) to sevuparin was measured. Flow Dynamic Adhesion: Rolling adhesion of isolated sickle-leukocytes on an L-selectin coated microfluidic channel was measured during physiologic flow. Isolated sickle-leukocytes were treated with increasing doses of sevuparin (0, 3, 7, 21, 200µg/mL) for 30 min. Dose response of rolling cell density (cells/mm2), rolling cell percentage (%), and average rolling velocity (µm/s) to sevuparin was assessed. Cell identification and tracking of rolling were digitally analyzed. Results:Statistically significant inhibition of sickle whole blood adhesion to HUVECs was observed at 3.0 µg/mL of sevuparin (p<0.001). In the same manner, statistically significant inhibition of sickle whole blood adhesion to VCAM-1 was observed at 200 µg/mL of sevuparin (p=0.033, absolute adhesion, p=0.001, % baseline adhesion). Each patient sample demonstrated a reduction in adhesion. Sevuparin also demonstrated a statistically significant dose-dependent reduction of sickle leukocyte rolling cell density (cells/mm2), rolling cell percentage (%), and an increase in average rolling velocity (µm/s) on L-selectin. Patient-to-patient variability in sevuparin response was observed. Conclusions: Sevuparin blocks both sickle whole blood and isolated sickle-leukocyte adhesive interactions under physiologic flow at clinically relevant concentrations. The blocking of adhesion to VCAM-1 indicates that sevuparin acts in the same manner as other heparinoids in vitro, and block the interaction with VLA-4. L-selectin is another possible target for sevuparin therapy now confirmed at the cellular level. Clinically, Okpala et al 2002 has shown that L-selectin expression by monocytes is increased in vaso-occlusive crises, compared to steady state and that both mononuclear cell and neutrophil L-selectin expression is also higher in patients with certain complications of SCD. Here we show that sevuparin acts in a multicellular manner, blocking both SS-RBC firm adhesion and L-selectin-mediated rolling adhesion of sickle-leukocytes, as well as functionally interacting with yet another key adhesion receptor VCAM-1. This further adds to sevuparin's multimodal action and its potential clinical benefits in treating the complex mechanisms manifested in vaso-occlusion and complications in SCD. Disclosures Lindgren: Dilaforette AB: Employment. White:Functional Fluidics: Employment, Equity Ownership. Liu:Functional Fluidics: Employment, Equity Ownership. Jendeberg:Dilaforette AB: Employment. Hines:Fucntional Fluidics: Employment, Equity Ownership.

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Inhibition of E-Selectin Inflammatory Function by the Glycomimetic GMI-1070

Blood ◽

10.1182/blood.v118.21.851.851 ◽

2011 ◽

Vol 118 (21) ◽

pp. 851-851

Author(s):

Scott I Simon ◽

Shannon Chase ◽

Helen Thackray ◽

John L. Magnani ◽

Ted Wun

Keyword(s):

Whole Blood ◽

Immune Surveillance ◽

Flow Chamber ◽

Cell Disease ◽

Integrin Activation ◽

Employment Equity ◽

Rolling Velocity ◽

Equity Ownership ◽

Slow Rolling ◽

Dose Dependent

Abstract Abstract 851FN2 Introduction: E-selectin expression by endothelium plays dual roles in inflammation by supporting slow rolling and subsequently eliciting integrin activation and arrest of leukocytes. This process may be important for immune surveillance. In a previous mouse model of sickle cell disease, E-selectin mediated outside-in signaling results in upregulated leukocyte Mac-1 and increased red cell capture, exacerbating vaso-occlusion. This process was attenuated by infusion of GMI-1070, a novel synthetic small molecule pan-selectin antagonist. GMI-1070 is now in Phase II clinical trial to determine efficacy in treatment of vaso-occlusive crisis (VOC) of sickle cell disease (SCD). Here, we studied its dose dependent effects in SCD subjects not in VOC on neutrophil activation and its effects on E-selectin mediated β2 integrin activation and rolling and arrest in shear flow. Methods: Samples were obtained from 4 SCD subjects not in VOC enrolled in a Phase I study of the effects of GMI-1070, a pan-selectin antagonist that preferentially inhibits E-selectin. An intravenous (IV) loading dose of 20 mg/kg was followed 10 hours later by a dose of 10 mg/kg. Samples were drawn before the loading dose, then 4 and 8 hours after the initial infusion. Polymorphonuclear Neutrophil (PMN) activation was analyzed in whole blood and isolated cell assays. Monoclonal antibodies and fluorescent-activated cell sorting (FACS) were used to assay expression of CD11b/CD18 (Mac-1), CD62L (L-selectin), and the high affinity active conformation of CD18 (327C) as markers of neutrophil activation. E-selectin mediated activation of CD18 was achieved in isolated PMN by incubating with E-selectin-IgG and goat antibody F(ab')2 fragment to crosslink E-selectin-IgG PMN activation was assessed by surface expression of high affinity CD18 by the mAb 327C and FACS in the presence of various concentrations of GMI-1070. PMN rolling and arrest on an inflammatory substrate was quantified using a lab on a chip assay. Whole blood or isolated PMN were perfused through a microfluidic flow chamber at a shear stress of 2 dynes/cm2. The flow chamber had immobilized E-selectin and ICAM-1 to support PMN rolling and adhesion. Video recordings of PMN interacting with this substrate were taken to quantify the number of rolling versus arrested cells and to measure rolling velocity in the presence of GMI-1070. Results: An inverse relationship between the serum concentration of GMI-1070 and either activated CD18 or upregulated CD11b was observed. In the flow chamber assays, 6 of 7 samples showed GMI-1070 diminished PMN arrest that also correlated with diminished integrin activation. Incubation of PMN with GMI-1070 blocked CD18 activation in response to E-selectin-IgG cross-linking, with an IC50 of 0.5 mM. PMN rolling and arrest was measured following shearing of isolated PMN on E-selectin and ICAM-1 using a lab on a chip assay. The mean rolling velocity of 2 mm/sec was increased to 6 mm/sec at a GMI-1070 concentration of 20 mM, with an IC50 of 5.5 mM for the increase in rolling velocity. In contrast, an IC50 = 0.8 mM was required to antagonize the fraction converting from rolling to arrest under shear flow. Summary and Conclusions: There was a dose dependent inhibitory effect on PMN activation after IV administration of GMI-1070 as measured in ex vivo whole blood samples in a small cohort of SCD subjects at steady state. A systematic study of the activity of GMI-1070 on isolated PMN revealed two distinct patterns of inhibition. At low concentrations (IC50 = 0.5mM), GMI-1070 effectively blocked the capacity of E-selectin to activate CD18, in response to cross-linked ligands, and mediate arrest. Only at higher concentrations (IC50 = 5.5mM) did we observe a significant alteration in the capacity of GMI-1070 to increase the rolling velocity and frequency of capture on a substrate of E-selectin under fluid shear stress. This differential capacity to alter function reveals that slow rolling, which is mediated by recognition of a number of sialylated ligands on the surface of PMN can be distinguished from the processes associated with the transition to arrest. Thus, treatment with GMI-1070 at doses that efficiently block vascular occlusion may spare some PMN rolling and immune surveillance function. Disclosures: Simon: GlycoMimetics Inc.: Research Funding. Chase:GlycoMimetics Inc.: Research Funding. Thackray:GlycoMimetics, Inc.: Employment, Equity Ownership. Magnani:GlycoMimetics, Inc.: Employment, Equity Ownership.

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CTP-221, a Deuterated S-Enantiomer of Lenalidomide, Possesses Significantly Enhanced Immunomodulatory and Anti-Proliferative Properties Relative to the R-Enantiomer and to Racemic Lenalidomide.

Blood ◽

10.1182/blood.v120.21.2463.2463 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2463-2463 ◽

Cited By ~ 3

Author(s):

Lijun Wu ◽

Ara M. Aslanian ◽

Julie F. Liu ◽

Kristine Hogan ◽

Roger Tung

Keyword(s):

Multiple Myeloma ◽

Clinical Efficacy ◽

Whole Blood ◽

Biological Activities ◽

Employment Equity ◽

Myeloma Cells ◽

Tnf Α ◽

Racemic Mixtures ◽

Equity Ownership

Abstract Abstract 2463 Lenalidomide (Revlimid®) is an immunomodulatory drug (IMiD) currently approved for the treatment of 5q- myelodysplastic syndrome and multiple myeloma. The clinical efficacy of lenalidomide is thought to be related in part to enhanced T-cell co-stimulation and NK-cell activation via augmented IL-2 and IFN-γ production (Bartlett et al., 2004; Corral and Kaplan, 1999). Lenalidomide also inhibits TNF-α production in peripheral blood mononuclear cells (PBMCs) and whole blood, which may further contribute to its anti-tumor activity (Mueller et al., 1999). In addition to immunomodulatory effects, lenalidomide directly induces growth arrest and apoptosis in multiple myeloma cells, which is also recognized as a key mechanism of clinical efficacy (Mitsiades, 2002; Bartlett et al., 2004). IMiD-class compounds, including thalidomide, lenalidomide, and pomalidomide, have been developed as racemic mixtures of S- and R-enantiomers. The isolated enantiomers of thalidomide are known to have distinct biological activities. For example, the well-documented sedative effects of thalidomide are correlated with the R-enantiomer (Eriksson et al., 2000), whereas S-thalidomide exhibits enhanced potency for TNF-α inhibition and IL-2 induction compared to R-thalidomide (Mueller et al., 1999; Moreira et al., 2003; Macor, 2007). Due to facile in vivo conversion, isolated S- enantiomers of IMiDs have not been developed clinically. To our knowledge, it has not been previously reported whether lenalidomide has enantiospecific immunomodulatory, anti-proliferative, or toxicological properties. Given the therapeutic importance of lenalidomide, we explored a number of deuterium-substituted analogs of lenalidomide, either as racemic mixtures or as isolated S- and R-enantiomers, and studied them in several in vitro pharmacological assays. We found that in each case tested, deuterated racemic lenalidomide analogs were indistinguishable from non-deuterated lenalidomide across all the assays employed, including IL-2 induction in anti-CD3-stimulated PBMC, TNF-α inhibition in LPS-stimulated whole blood, and inhibition of proliferation of MM.1S human multiple myeloma cells. In contrast to deuterated racemic lenalidomide, CTP-221, an optimized deuterated S-lenalidomide analog, exhibited enhanced potency compared to racemic lenalidomide for IL-2 induction (2.7-fold), TNF-α inhibition (3.7-fold) and anti-proliferative (2.4-fold) activities in vitro. Interestingly, these enhancements in potency are greater than the maximal 2-fold enhancement one could expect from assessing an isolated active enantiomer in comparison to its racemate. These greater-than-expected enhancements in potency were consistently observed across all the assays comparing CTP-221 to lenalidomide, suggesting that deuterium substitution had additional effect(s) that drive increased potency. Furthermore, CTP-221 was significantly more potent than similarly deuterated R-lenalidomide in these assays (between 9.0 and 19.8-fold), demonstrating that the clinically relevant pharmacological activities of lenalidomide are primarily contained within the S-enantiomer. Finally, we found that CTP-221 was consistently more potent (1.2–2.0-fold) than non-deuterated S-lenalidomide. Taken together, these in vitro data demonstrate that deuterated racemic lenalidomide does not offer apparent advantages versus lenalidomide. However, the deuterated S-lenalidomide analog CTP-221 is significantly more potent than lenalidomide in key biological activities believed important for clinical efficacy. We plan to explore the toxicological properties of CTP-221 to assess its therapeutic window relative to lenalidomide. Disclosures: Wu: Concert Pharmaceuticals, Inc.: Employment, Equity Ownership. Aslanian:Concert Pharmaceuticals, Inc.: Employment, Equity Ownership. Liu:Concert Pharmaceuticals, Inc.: Employment, Equity Ownership. Hogan:Concert Pharmaceuticals, Inc.: Employment, Equity Ownership. Tung:Concert Pharmaceuticals, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

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Single Cell-Level Signaling Profiling of Acute Myeloid Leukemia Following Treatment with Axl Kinase Inhibitor BGB324

Blood ◽

10.1182/blood.v126.23.4931.4931 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4931-4931

Author(s):

Monica Hellesøy ◽

Katarzyna Wnuk-Lipinska ◽

Anna Boniecka ◽

Eline Milde Nævdal ◽

Hakon Reikvam ◽

...

Keyword(s):

Signal Transduction ◽

Overall Survival ◽

Cell Line ◽

Research Funding ◽

Xenograft Model ◽

Employment Equity ◽

In Vitro Response ◽

Equity Ownership ◽

Dose Dependent

Abstract Axl is a member of the Tyro3, Axl, Mer (TAM) receptor tyrosine kinase family that regulate a wide range of cellular functions, including cell survival, proliferation, migration/invasion and adhesion. Axl has been shown to play a key role in the survival and metastasis of many tumors, and has also been found to be upregulated and constitutively active in human AML. Indeed, Axl has been reported as an independent prognostic marker and a potential novel therapeutic target in AML. BGB324 is a first-in-class highly selective small molecule inhibitor of Axl. BGB324 has been shown to be safe and well tolerated in clinical safety studies in healthy volunteers at doses up to 1500 mg/day with a predictable PK profile and long plasma half-life, and is currently in phase I b clinical trials for AML and non-small cell lung cancer. In this study, we use phosphoflow cytometry to measure changes in signal transduction nodes in single AML cells treated with BGB324. We are applying this approach to monitor signaling profiles in primary AML cells harvested from patients undergoing BGB324 treatment. Results: The human AML cell line MOLM13 was treated in vitro with BGB324 (0.5 and 1µM for 1 hour) and analyzed for signal transduction changes by phosphoflow cytometry. We found a significant reduction in phosphorylation of Axl (pY779), Akt(pS473), Erk1/2(pT202/Y204) and PLCɣ1(pY783). Next we established a systemic MOLM13 preclinical AML model in NOD/SCID mice. The mice were treated with 25 or 50 mg/kg BGB324 until moribund (up to 16 days). We found a dose-dependent and significant increase in overall survival in BGB324-treated mice. We further investigated intracellular signaling in BGB324-treated cells in vivo. Mice carrying systemic AML disease (MOLM13) were treated with BGB324 at 50mg/kg for 4 days, and we monitored CD33/45-positive MOLM13 cells harvested from spleen and bone marrow by flow cytometry. BGB324-treated mice showed a significant reduction in pErk and pPLCɣ1 relative to mice in the control group. PBMCs from peripheral blood of AML patients treated with BGB324 400 mg x1 at day 1 and 2, and thereafter 100 mg daily were collected for single cell signal profiling of signal transduction changes by conventional flow cytometry (phospho-flow) and mass cytometry (CyTOF). Preliminary phopho-flow analyses show decrease of pAkt(T308) and pPLCgamma1(Y783) in one patient. Further analyses are ongoing and will be presented. Figure 1. In vitro response to 1 hour BGB324 treatment in human AML cell line MOLM13 at 0.5 and 1µM doses. Response was evaluated in pAxl, pErk1/2, pAkt and pPLCγ1. n=3, *p≤0.05, **p≤0.005. Figure 1. In vitro response to 1 hour BGB324 treatment in human AML cell line MOLM13 at 0.5 and 1µM doses. Response was evaluated in pAxl, pErk1/2, pAkt and pPLCγ1. n=3, *p≤0.05, **p≤0.005. Figure 2. Dose-dependent response in overall survival in a MOLM13 systemic xenograft model (n=10). Figure 2. Dose-dependent response in overall survival in a MOLM13 systemic xenograft model (n=10). Figure 3. Response to BGB324-treatment in pErk, pPLCγ1 and pAkt in CD33/CD45-positive cells harvested from spleens (left) and bone marrows (right) of mice with systemic MOLM13 xenografts. n=5, *p≤0.05, **p≤0.005. Figure 3. Response to BGB324-treatment in pErk, pPLCγ1 and pAkt in CD33/CD45-positive cells harvested from spleens (left) and bone marrows (right) of mice with systemic MOLM13 xenografts. n=5, *p≤0.05, **p≤0.005. Disclosures Hellesøy: BerGenBio AS: Other: Previous employee. Stock option holder. Wnuk-Lipinska:BerGenBio AS: Employment. Boniecka:BerGenBio AS: Employment. Nævdal:BerGenBio AS: Employment. Loges:BerGenBio: Honoraria, Other: travel support, Research Funding. Cortes:Teva: Research Funding; BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; BerGenBio AS: Research Funding; Ariad: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Ambit: Consultancy, Research Funding; Arog: Research Funding; Celator: Research Funding; Jenssen: Consultancy. Lorens:BerGenBio AS: Employment, Equity Ownership. Micklem:BerGenBio AS: Employment, Equity Ownership. Gausdal:BerGenBio AS: Employment. Gjertsen:Haukeland University Hospital: Research Funding.

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Single Cell Network Profiling (SCNP) to Evaluate the Mechanism of Action of ON 01910.Na, A Novel Clinical Trial Stage Compound.

Blood ◽

10.1182/blood.v114.22.3827.3827 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3827-3827 ◽

Cited By ~ 2

Author(s):

David M. Soper ◽

Ying-Wen Huang ◽

Francois Wilhelm ◽

S. C. Cosenza ◽

E. Premkumar Reddy ◽

...

Keyword(s):

Cell Cycle ◽

Dose Range ◽

Histone H3 ◽

Cyclin B1 ◽

Employment Equity ◽

Cell Network ◽

M Phase ◽

Equity Ownership ◽

Dose Dependent

Abstract Abstract 3827 Poster Board III-763 Background ON 01910.Na, a small molecule multikinase inhibitor, promotes G2/M arrest and apoptosis. Key targets for this inhibitor include Plk1 (polo-like kinase, a cell cycle regulator), Cdk1, (cyclin dependent kinase, a mitotic regulator) and the PI-3 kinase pathway (Ramana Reddy et al. J. Med. Chem. 2008, Park et al, Oncogene, 2007, Gumireddy et al., Cancer Cell, 2005). The drug has been shown to have anti-tumor activity in in vitro and in vivo models. Phase I studies in >100 advanced cancer patients revealed that the drug is well tolerated. Further, in several ongoing Phase 1 clinical trials in patients with myelodysplastic syndromes (MDS), positive effects on hematological indicators have been noted (Sloand et al, ASH 2008). Based on these data, a Phase 2 single-arm study is in progress to assess the efficacy and safety of the drug in IPSS Intermediate-2 and High risk MDS patients. Single Cell Network Profiling (SCNP) using flow cytometry is a platform that measures multiple fluorescent parameters (up to 10) in each cell, including both surface markers and intracellular signaling proteins in response to extracellular network inputs. By simultaneously measuring the effects of drug exposure on several pathways within each cell type in a heterogeneous patient tissue sample, valuable data can be gained about drug interactions with specific cellular pathways and cell type selectivity. This information has potential implications for dose/schedule optimization and development of patient stratification biomarkers. Objectives Studies were designed to evaluate the in vitro effects of ON 01910.Na, at clinically relevant concentrations, on intracellular pathways in the human GM-CSF-dependent erythroblastic TF-1 cell line using SCNP in order to monitor transitional changes in the cell cycle, with a focus on the G2-M phase and to perform dose-dependent titrations of drug using these cell cycle readouts. Methods The reagents chosen to measure cell cycle readouts were fluorochrome-conjugated antibodies that recognize cyclin B1, p-histone H3(S28) and p-Cdk1(Y15) and 4'6'-diamino-2-phenylindole (DAPI), a fluorescent dye that binds strongly to DNA. The phosphorylation status of p-histone H3(S28) and p-Cdk1(Y15), and the level of cyclin B1 expression are all determinants of the G2-M and/or M phase of the cell cycle. Dose dependent titrations of ON 01910.Na and its inactive analog ON 01911 were performed over a dose range starting at 10-5 M and decreasing to 10-10 M (dose range which includes pharmacologically achievable concentrations in humans) with 3-fold serial dilutions for eleven points after an exposure to the drug for either 24 or 48 hrs. Cells were processed for multiparameter flow cytometry by fixation, permeabilization and incubation with fluorchrome-conjugated antibodies. Results The data showed that at 24 hours after ON 01910.Na exposure there was a simultaneous increase in phosphorylation of histone H3(S28), a decrease in phosphorylation of Cdk-1(Y15), and accumulation of cyclin B1. These data suggest that ON 01910 exposure disrupted the G2/M cell cycle transition leading to mitotic arrest with subsequent apoptosis. TF-1 cell DNA content measured by DAPI verified this to be the case as increases in G2/M and sub-G1 (a measure of apoptotic cell death) were simultaneously observed. No significant effects on G2/M targets were observed when TF-1 cells were exposed to ON 01911, indicating the effects of ON 01910.Na on the cell cycle were specific to the drug. Maximal effects of ON-01910.Na on cell cycle signaling molecules were observed at a drug concentration of 0.37 mM and no further changes were seen at higher concentrations. These effects were also observed at 48 hours, although with more cell death. Conclusions These data indicate that intracellular phosphorylation changes of histone H3(S28) and Cdk-1(Y15), in addition to accumulation of cyclin B1 with subsequent apoptosis, reflect possible mechanisms of action of ON 01910.Na. The assay will be used in ongoing clinical trials to measure the pharmacodynamic activity of the drug in MDS patient samples pre- and post-treatment. Disclosures: Soper: Nodality Inc.: Employment, Equity Ownership. Huang:Nodality Inc.: Employment, Equity Ownership. Wilhelm:Onconova Therapeutics Inc: Employment. Cosenza:Onconova Therapeutics Inc.: Consultancy. Reddy:Onconova Therapeutics Inc.: Consultancy, Equity Ownership, Grantee, Membership on an entity's Board of Directors or advisory committees. Cesano:Nodality Inc.: Employment, Equity Ownership. Greenberg:Nodality Inc.: Research Funding; Onconova Therapeutics Inc.: Research Funding. Fantl:Nodality, Inc.: Employment, Equity Ownership.

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M281: A Therapeutic Anti-FcRn Blocking Antibody for Rapid Clearance of IgG and IgG Autoantibodies in Immune Cytopenias and Other Auto/Allo-Immune Disease

Blood ◽

10.1182/blood.v126.23.3472.3472 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3472-3472 ◽

Cited By ~ 2

Author(s):

Leona E Ling ◽

Sucharita Roy ◽

Thomas Daly ◽

Edward Cochran ◽

Steven Tyler ◽

...

Keyword(s):

Endothelial Cells ◽

Umbilical Vein ◽

Recombinant Antibody ◽

In Vivo Studies ◽

Employment Equity ◽

Equity Ownership ◽

Models Of Disease ◽

Dose Dependent

Abstract Introduction IgG antibodies are the primary pathogenic agent in a number of auto- or allo-immune diseases. Efficacious therapies which decrease systemic levels of pathogenic antibodies include treatment with IVIG therapeutic plasmapheresis or immunoabsorption. Here, a novel strategy was evaluated to induce IgG clearance in diseases driven by IgG autoantibodies by blockade of FcRn-mediated IgG recycling. Methods M281 was developed as a high affinity, effectorless IgG1anti-FcRn monoclonal antibody. M281 effect on IgG recycling was evaluated in human umbilical vein endothelial cells in vitro. In vivo studies in transgenic human FCGRT/mouse FCGRT null mice and cynomolgus monkey were performed to characterize the pharmacokinetics, biodistribution, target occupancy, specificity of M281 and its efficacy in mouse models of thrombocytopenia and arthritis. Results M281 demonstrates specific dose-dependent, albumin-sparing IgG clearance in human FCGRT transgenic/mouse FCGRT null mice and in cynomolgus monkeys. M281 inhibits IgG recycling in endothelial cells in vitro and IgG clearance in vivo. Pharmacokinetics, target occupancy, pharmacodynamics and biodistribution indicate typical recombinant antibody biodistribution with rapid, dose dependent target inhibition and systemic clearance. M281 also demonstrated efficacy in mouse idiopathic thrombocytopenia purpura and collagen antibody-induced arthritis models of disease. Conclusions These findings support the evaluation of M281 as a strategy for the rapid and reversible suppression of pathogenic autoantibodies or alloantibodies in the setting of immune cytopenias, acquired inhibitors, thrombotic states and other disorders. Disclosures Ling: Momenta Pharmaceuticals: Employment, Equity Ownership. Roy:Momenta Pharmaceuticals: Employment, Equity Ownership. Daly:Momenta Pharmaceuticals: Employment, Equity Ownership. Cochran:Momenta Pharmaceuticals: Employment, Equity Ownership. Tyler:Momenta Pharmaceuticals: Employment, Equity Ownership. Markowitz:Momenta Pharmaceuticals: Employment, Equity Ownership. Bulik:Momenta Pharmaceuticals: Employment, Equity Ownership. Choudhury:Momenta Pharmaceuticals: Employment, Equity Ownership. Meador:Momenta Pharmaceuticals: Employment, Equity Ownership. Parge:Momenta Pharmaceuticals: Employment. Mekala:Momenta Pharmaceuticals: Employment, Equity Ownership. Sipsey:Momenta Pharmaceuticals: Employment, Equity Ownership. Gurnani:Momenta Pharmaceuticals: Employment, Equity Ownership. Duffner:Momenta Pharmaceuticals: Employment, Equity Ownership. Lee:Momenta Pharmaceuticals: Employment, Equity Ownership. Washburn:Momenta Pharmaceuticals: Employment, Equity Ownership. Meccariello:Momenta Pharmaceuticals: Employment, Equity Ownership. Schaeck:Momenta Pharmaceuticals: Employment, Equity Ownership. Wang:Momenta Pharmaceuticals: Employment, Equity Ownership. Schultes:Momenta Pharmaceuticals: Employment, Equity Ownership. Hillson:Momenta Pharmaceuticals: Employment, Equity Ownership. Avery:Momenta Pharmaceuticals: Employment, Equity Ownership. Kaundinya:Momenta Pharmaceuticals: Employment, Equity Ownership. Manning:Momenta Pharmaceuticals: Employment, Equity Ownership.

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Elucidating the Excessive Pro-Coagulant Effect of a Sequence Identical Analogue to ACE910 in Combination with Bypassing Agents

Blood ◽

10.1182/blood.v130.suppl_1.90.90 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 90-90

Author(s):

Rudolf Hartmann ◽

Tjerk Feenstra ◽

Sabine Knappe ◽

Michael Dockal ◽

Friedrich Scheiflinger

Keyword(s):

Whole Blood ◽

Thrombin Generation ◽

Hemophilia A ◽

Thrombus Formation ◽

Phase Iii ◽

Hek293 Cells ◽

Employment Equity ◽

Equity Ownership ◽

Bypassing Agents

Abstract Introduction: Emicizumab (ACE910), an antibody to FIX(a) and FX(a), is currently under investigation for treatment of hemophilia with inhibitors. In a phase III trial, two thromboembolic complications and three cases of microangiopathy were reported in patients on ACE910 prophylaxis [Oldenburg et al. NEJM 2017], whose breakthrough bleeding was treated with activated prothrombin complex concentrate aPCC (FEIBA) or aPCC and rFVIIa. We generated a sequence identical analogue (SIA) to ACE910 and analyzed its synergistic interplay with bypassing agents. Aims: To monitor in vitro the pro-coagulant activity of SIA ACE910 in the presence of FEIBA and rFVIIa, and detect the source of excessive coagulation induced by SIA ACE910 combined with FEIBA. Methods: A sequence identical analogue (SIA) to ACE910 was expressed in HEK293 cells, purified as previously described [Sampei et al. PLoS One 2013], and analyzed in several global hemostatic assays at different concentrations and test conditions using plasma and whole blood assays. In thrombin generation (TG) experiments, platelet-poor plasma (PPP) from hemophilia A inhibitor patients and hemophilia A plasma reconstituted with platelets from 3 healthy donors (PRP) was used. A normal TG range was established in healthy donor plasma. Therapeutic concentrations of SIA ACE910 (20-600 nM) were tested alone and with FEIBA (0.05-1 U/mL) or rFVIIa (0.88-5.25 µg/mL). To measure FEIBA components' contribution to the synergistic effect with SIA ACE910, PPP was spiked with select FEIBA components at concentrations corresponding to 0.5 U/mL FEIBA in combination with the antibody. Thrombus formation was analyzed in FVIII-inhibited blood using rotational thromboelastometry (ROTEM) and Total Thrombus-formation Analysis System (T-TAS). Results: Normal peak thrombin was 47-144 nM for PPP and 88-231 nM for PRP. rFVIIa and FEIBA had an additive effect on TG in combination with SIA ACE910 in both plasma types. Combined with rFVIIa (0.88 µg/mL) or FEIBA (0.5 U/mL), SIA ACE910 (600 nM) induced a ~2- and ~16-fold increase over SIA ACE910 alone. SIA ACE910+rFVIIa did not reach the normal range, while SIA ACE910+FEIBA far exceeded it. Adding individual FEIBA components to PPP showed that FIX was, with a half-maximal effect, the main driver for enhanced TG, followed by FIXa. formation in FVIII-inhibited whole blood using ROTEM and T-TAS confirmed the excessive effect of SIA ACE910+FEIBA. In ROTEM, FEIBA and rFVIIa reduced clotting time to shorter than normal, whereas SIA ACE910 had only little effect. Moreover, adding SIA ACE910 to rFVIIa exerted no effect over rFVIIa alone. Conclusion: Combining SIA ACE910 at plasma concentrations observed in patients [Oldenburg et al. NEJM 2017] with FEIBA induced excessive thrombin generation and faster clot formation. In vitro, this effect is mainly mediated by FEIBA component FIX. ACE910 binds to FIX and FIXa to the same extent, and displays its pro-coagulant effect via an unregulated mechanism. Therefore, careful judgement is needed in treating breakthrough bleeds with FEIBA. Disclosures Hartmann: Shire: Employment. Feenstra: Shire: Employment. Knappe: Shire: Employment. Dockal: Baxalta: Patents & Royalties; Shire: Employment, Equity Ownership; Baxter: Equity Ownership, Patents & Royalties. Scheiflinger: Baxter: Equity Ownership; Shire: Employment, Equity Ownership.

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Dipyridamole Inhibits Platelet Aggregation in Whole Blood

Thrombosis and Haemostasis ◽

10.1055/s-0038-1665327 ◽

1983 ◽

Vol 50 (04) ◽

pp. 852-856 ◽

Cited By ~ 104

Author(s):

P Gresele ◽

C Zoja ◽

H Deckmyn ◽

J Arnout ◽

J Vermylen ◽

...

Keyword(s):

Platelet Aggregation ◽

Whole Blood ◽

Ex Vivo ◽

Platelet Rich Plasma ◽

Significant Negative Correlation ◽

Significant Inhibition ◽

Oral Drug ◽

Human Blood Samples ◽

Induced Aggregation

SummaryDipyridamole possesses antithrombotic properties in the animal and in man but it does not inhibit platelet aggregation in plasma. We evaluated the effect of dipyridamole ex vivo and in vitro on platelet aggregation induced by collagen and adenosine- 5’-diphosphate (ADP) in human whole blood with an impedance aggregometer. Two hundred mg dipyridamole induced a significant inhibition of both ADP- and collagen-induced aggregation in human blood samples taken 2 hr after oral drug intake. Administration of the drug for four days, 400 mg/day, further increased the antiplatelet effect. A significant negative correlation was found between collagen-induced platelet aggregation in whole blood and dipyridamole levels in plasma (p <0.001). A statistically significant inhibition of both collagen (p <0.0025) and ADP-induced (p <0.005) platelet aggregation was also obtained by incubating whole blood in vitro for 2 min at 37° C with dipyridamole (3.9 μM). No such effects were seen in platelet-rich plasma, even after enrichment with leukocytes. Low-dose adenosine enhanced in vitro inhibition in whole blood.Our results demonstrate that dipyridamole impedes platelet aggregation in whole blood by an interaction with red blood cells, probably involving adenosine.

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THU0080 PRECLINICAL CHARACTERIZATION OF TLL018, A NOVEL, HIGHLY POTENT AND SELECTIVE JAK1/TYK2 INHIBITOR FOR TREATING AUTOIMMUNE DISEASES

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2020-eular.1547 ◽

2020 ◽

Vol 79 (Suppl 1) ◽

pp. 252.1-252

Author(s):

X. Liu ◽

F. Tan ◽

C. Liang

Keyword(s):

Bone Resorption ◽

Autoimmune Diseases ◽

Whole Blood ◽

Significant Inhibition ◽

Clinical Development ◽

Therapeutic Window ◽

Collagen Induced Arthritis

Background:Janus kinases (JAKs) are important regulators of intracellular responses triggered by many key proinflammatory cytokines and are clinically validated therapeutic targets for treating various autoimmune diseases. However, current approved JAK inhibitors failed to achieve maximal clinical benefit in part due to their unfavorable selectivity for individual JAKs such as JAK2 and/or JAK3, leading to dose-limiting toxicities or severe toxicities (e.g., thrombosis, anemia, immune suppression). Selective inhibition of JAK1 and/or TYK2 may minimize or avoid some of the toxicities and potentially offer a better therapeutic window for treating autoimmune diseases. No highly selective JAK1/TYK2 inhibitor has been reported to date.Objectives:Discovery of a highly selective JAK1/TYK2 inhibitor that maximally avoids JAK2 and JAK3 inhibition. We described preclinical characterization of a novel, highly potent and selective JAK1/TYK2 inhibitor TLL018 and its potential utility in treating autoimmune diseases such as rheumatoid arthritis (RA).Methods:Using predicting SAR, TLL018 was designed to achieve exquisite selectivity for both JAK1 and TYK2 while sparing JAK2, JAK3 and other human kinases. Its enzyme and cell activities, kinase selectivity, andin vivoefficacy were assessed in a battery of relevant enzyme, cell and whole blood assays, andin vivoarthritis animal models. Additional preclinical DMPK and toxicology studies were conducted to support its clinical development.Results:TLL018 is a highly potent and selective, orally bioavailable JAK1/TYK2 inhibitor against JAK1 (IC50= 4 nM) and TYK2 (IC50= 5 nM) as measured inin vitrokinase assays with ATP concentrations at individual Km. Its potency against JAK2 or JAK3 is greater than 1 µM. Profiling against a panel of over 350 human kinase showed that TLL018 is exclusively selective for JAK1 and TYK2, with ≥ 90-fold selectivity against all other kinases tested. TLL018 exhibited potent cellular activity for JAK1-mediated IL-6 signaling (IC50= 0.6 µM) with greater than 100-fold selectivity against JAK2-mediated cytokine (e.g., TPO) signaling in human whole blood-based assays.Oral administration of TLL018 demonstrated dose-dependent efficacy in commonly studied rat adjuvant-induced arthritis (rAIA) model and mouse collagen-induced arthritis (mCIA) model. Significant inhibition of inflammation, bone resorption, splenomegaly and body weight change was observed in adjuvant-induced disease in rats. In addition, significant inhibition of inflammation, cartilage destruction, bone resorption and histological signs was demonstrated in collagen-induced arthritis in mice. Noticeably, TLL018 exhibited significant anti-inflammation activity at doses that only blocked JAK1 and TYK2 and exerted little inhibition of JAK2 and JAK3.In support of clinical development of TLL018, preclinical ADME and PK studies and IND-enabling toxicology and safety pharmacology studies were completed, confirming that TLL018 possesses excellent ADME and PK properties, and exhibits a clean on-target safety profile.Conclusion:TLL018 is a highly potent and selective JAK1/TYK2 inhibitor that demonstrated excellent efficacy and tolerability in relevant mouse and rat arthritis models. The collective data of its preclinical pharmacology, PK and toxicology showed a favorable pharmaceutical profile, further supporting its development for treating autoimmune diseases including RA. Clinical evaluation of TLL018 is ongoing.Disclosure of Interests:Xiangdong Liu Shareholder of: I own shares of TLL Pharmaceutical LLC, Employee of: I am employed by TLL Pharmaceutical LLC, Fenlai Tan Shareholder of: I own shares of TLL Pharmaceutical LLC, Employee of: I am employed by TLL Pharmaceutical LLC, Chris Liang Shareholder of: I own shares of TLL Pharmaceutical LLC, Employee of: I am employed by TLL Pharmaceutical LLC

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Phenotype Does Not Always Equal Function: HDAC Inhibitors and UM171, but Not SR1, Lead to Rapid Upregulation of CD90 on Non-Engrafting CD34+CD90-Negative Human Cells

Blood ◽

10.1182/blood.v130.suppl_1.659.659 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 659-659

Author(s):

Kevin A. Goncalves ◽

Megan D. Hoban ◽

Jennifer L. Proctor ◽

Hillary L. Adams ◽

Sharon L. Hyzy ◽

...

Keyword(s):

In Vitro Culture ◽

Small Molecule ◽

Hdac Inhibitors ◽

Employment Equity ◽

Nsg Mice ◽

Cd34 Cells ◽

Equity Ownership ◽

Human Hscs

Abstract Background. The ability to expand human hematopoietic stem cells (HSCs) has the potential to improve outcomes in HSC transplantation and increase the dose of gene-modified HSCs. While many approaches have been reported to expand HSCs, a direct comparison of the various methods to expand transplantable HSCs has not been published and clinical outcome data for the various methods is incomplete. In the present study, we compared several small molecule approaches reported to expand human HSCs including HDAC inhibitors, the aryl hydrocarbon antagonist, SR1, and UM171, a small molecule with unknown mechanism, for the ability to expand phenotypic HSC during in vitro culture and to expand cells that engraft NSG mice. Although all strategies increased the number of phenotypic HSC (CD34+CD90+CD45RA-) in vitro, SR1 was the most effective method to increase the number of NOD-SCID engrafting cells. Importantly, we found that HDAC inhibitors and UM171 upregulated phenotypic stem cell markers on downstream progenitors, suggesting that these compounds do not expand true HSCs. Methods. Small-molecules, SR1, HDAC inhibitors (BG45, CAY10398, CAY10433, CAY10603, Entinostat, HC Toxin, LMK235, PCI-34051, Pyroxamide, Romidepsin, SAHA, Scriptaid, TMP269, Trichostatin A, or Valproic Acid) and UM171 were titrated and then evaluated at their optimal concentrations in the presence of cytokines (TPO, SCF, FLT3L, and IL6) for the ability to expand human mobilized peripheral blood (mPB)-derived CD34+ cells ex vivo . Immunophenotype and cell numbers were assessed by flow cytometry following a 7-day expansion assay in 10-point dose-response (10 µM to 0.5 nM). HSC function was evaluated by enumeration of colony forming units in methylcellulose and a subset of the compounds were evaluated by transplanting expanded cells into sub-lethally irradiated NSG mice to assess engraftment potential in vivo . All cells expanded with compounds were compared to uncultured or vehicle-cultured cells. Results. Following 7 days of expansion, SR1 (5-fold), UM171 (4-fold), or HDAC inhibitors (>3-35-fold) resulted in an increase in CD34+CD90+CD45RA- number relative to cells cultured with cytokines alone; however, only SR1 (18-fold) and UM171 (8-fold) demonstrated enhanced engraftment in NSG mice. Interestingly, while HDAC inhibitors and UM171 gave the most robust increase in the number and frequency of CD34+CD90+CD45RA- cells during in vitro culture, these methods were inferior to SR1 at increasing NSG engrafting cells. The increase in CD34+CD90+CD45RA- cells observed during in vitro culture suggested that these compounds may be generating a false phenotype by upregulating CD90 and down-regulating CD45RA on progenitors that were originally CD34+CD90-CD45RA+. We tested this hypothesis by sorting CD34+CD90-CD45RA+ cells and culturing these with the various compounds. These experiments confirmed that both HDAC inhibitors (33-100 fold) and UM171 (28-fold) led to upregulation of CD90 on CD34+CD90-CD45RA+ cells after 4 days in culture. Since approximately 90% of the starting CD34+ cells were CD90-, these data suggest that most of the CD34+CD90+CD45RA- cells in cultures with HDAC inhibitors and UM171 arise from upregulation of CD90 rather than expansion of true CD34+CD90+CD45RA- cells and may explain the disconnect between in vitro HSC phenotype and NSG engraftment in vivo . This was further confirmed by evaluation of colony forming unit frequency of CD34+CD90-CD45RA+ cells after culture with compounds. Conclusions. We have showed that AHR antagonism is optimal for expanding functional human HSCs using the NSG engraftment model. We also demonstrated that UM171 and HDAC inhibitors upregulate phenotypic HSC markers on downstream progenitors. This could explain the discrepancy between impressive in vitro phenotypic expansion and insufficient functional activity in the NSG mouse model. Therefore, these data suggest caution when interpreting in vitro expansion phenotypes without confirmatory functional transplantation data, especially as these approaches move into clinical trials in patients. Disclosures Goncalves: Magenta Therapeutics: Employment, Equity Ownership. Hoban: Magenta Therapeutics: Employment, Equity Ownership. Proctor: Magenta Therapeutics: Employment, Equity Ownership. Adams: Magenta Therapeutics: Employment, Equity Ownership. Hyzy: Magenta Therapeutics: Employment, Equity Ownership. Boitano: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

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Highly Differentiated Anti-CD47 Antibody, AO-176, Potently Inhibits Hematologic Malignancies Alone and in Combination

Blood ◽

10.1182/blood-2019-126298 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1844-1844

Author(s):

John Richards ◽

Myriam N Bouchlaka ◽

Robyn J Puro ◽

Ben J Capoccia ◽

Ronald R Hiebsch ◽

...

Keyword(s):

Multiple Myeloma ◽

Combination Therapy ◽

Tumor Growth ◽

Growth Inhibition ◽

Tumor Cells ◽

Tumor Growth Inhibition ◽

Employment Equity ◽

Equity Ownership

AO-176 is a highly differentiated, humanized anti-CD47 IgG2 antibody that is unique among agents in this class of checkpoint inhibitors. AO-176 works by blocking the "don't eat me" signal, the standard mechanism of anti-CD47 antibodies, but also by directly killing tumor cells. Importantly, AO-176 binds preferentially to tumor cells, compared to normal cells, and binds even more potently to tumors in their acidic microenvironment (low pH). Hematological neoplasms are the fourth most frequently diagnosed cancers in both men and women and account for approximately 10% of all cancers. Here we describe AO-176, a highly differentiated anti-CD47 antibody that potently targets hematologic cancers in vitro and in vivo. As a single agent, AO-176 not only promotes phagocytosis (15-45%, EC50 = 0.33-4.1 µg/ml) of hematologic tumor cell lines (acute myeloid leukemia, non-Hodgkin's lymphoma, multiple myeloma, and T cell leukemia) but also directly targets and kills tumor cells (18-46% Annexin V positivity, EC50 = 0.63-10 µg/ml) in a non-ADCC manner. In combination with agents targeting CD20 (rituximab) or CD38 (daratumumab), AO-176 mediates enhanced phagocytosis of lymphoma and multiple myeloma cell lines, respectively. In vivo, AO-176 mediates potent monotherapy tumor growth inhibition of hematologic tumors including Raji B cell lymphoma and RPMI-8226 multiple myeloma xenograft models in a dose-dependent manner. Concomitant with tumor growth inhibition, immune cell infiltrates were observed with elevated numbers of macrophage and dendritic cells, along with increased pro-inflammatory cytokine levels in AO-176 treated animals. When combined with bortezomib, AO-176 was able to elicit complete tumor regression (100% CR in 10/10 animals treated with either 10 or 25 mg/kg AO-176 + 1 mg/kg bortezomib) with no detectable tumor out to 100 days at study termination. Overall survival was also greatly improved following combination therapy compared to animals treated with bortezomib or AO-176 alone. These data show that AO-176 exhibits promising monotherapy and combination therapy activity, both in vitro and in vivo, against hematologic cancers. These findings also add to the previously reported anti-tumor efficacy exhibited by AO-176 in solid tumor xenografts representing ovarian, gastric and breast cancer. With AO-176's highly differentiated MOA and binding characteristics, it may have the potential to improve upon the safety and efficacy profiles relative to other agents in this class. AO-176 is currently being evaluated in a Phase 1 clinical trial (NCT03834948) for the treatment of patients with select solid tumors. Disclosures Richards: Arch Oncology Inc.: Employment, Equity Ownership, Other: Salary. Bouchlaka:Arch Oncology Inc.: Consultancy, Equity Ownership. Puro:Arch Oncology Inc.: Employment, Equity Ownership. Capoccia:Arch Oncology Inc.: Employment, Equity Ownership. Hiebsch:Arch Oncology Inc.: Employment, Equity Ownership. Donio:Arch Oncology Inc.: Employment, Equity Ownership. Wilson:Arch Oncology Inc.: Employment, Equity Ownership. Chakraborty:Arch Oncology Inc.: Employment, Equity Ownership. Sung:Arch Oncology Inc.: Employment, Equity Ownership. Pereira:Arch Oncology Inc.: Employment, Equity Ownership.

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