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Sickle cell disease (SCD) is associated with hemolysis, vascular inflammation, and organ damage. Affected patients experience chronic painful vaso-occlusive events requiring hospitalization. Hypoxia-induced polymerization of sickle hemoglobin S (HbS) contributes to sickling of red blood cells (RBCs) and disease pathophysiology. Dilution of HbS with nonsickling hemoglobin or hemoglobin with increased oxygen affinity, such as fetal hemoglobin or HbS bound to aromatic aldehydes, is clinically beneficial in decreasing polymerization. We investigated a novel alternate approach to modify HbS and decrease polymerization by inhibiting methionine aminopeptidase 2 (MetAP2), which cleaves the initiator methionine (iMet) from Val1 of α-globin and βS-globin. Kinetic studies with MetAP2 show that βS-globin is a fivefold better substrate than α-globin. Knockdown of MetAP2 in human umbilical cord blood–derived erythroid progenitor 2 cells shows more extensive modification of α-globin than β-globin, consistent with kinetic data. Treatment of human erythroid cells in vitro or Townes SCD mice in vivo with selective MetAP2 inhibitors extensively modifies both globins with N-terminal iMet and acetylated iMet. HbS modification by MetAP2 inhibition increases oxygen affinity, as measured by decreased oxygen tension at which hemoglobin is 50% saturated. Acetyl-iMet modification on βS-globin delays HbS polymerization under hypoxia. MetAP2 inhibitor–treated Townes mice reach 50% total HbS modification, significantly increasing the affinity of RBCs for oxygen, increasing whole blood single-cell RBC oxygen saturation, and decreasing fractional flow velocity losses in blood rheology under decreased oxygen pressures. Crystal structures of modified HbS variants show stabilization of the nonpolymerizing high O2–affinity R2 state, explaining modified HbS antisickling activity. Further study of MetAP2 inhibition as a potential therapeutic target for SCD is warranted.

Sickle cell disease (SCD) is associated with hemolysis, vascular inflammation, and organ damage. Affected patients experience chronic painful vaso-occlusive events requiring hospitalization. Hypoxia-induced polymerization of sickle hemoglobin S (HbS) contributes to sickling of red blood cells (RBCs) and disease pathophysiology. Dilution of HbS with nonsickling hemoglobin or hemoglobin with increased oxygen affinity, such as fetal hemoglobin or HbS bound to aromatic aldehydes, is clinically beneficial in decreasing polymerization. We investigated a novel alternate approach to modify HbS and decrease polymerization by inhibiting methionine aminopeptidase 2 (MetAP2), which cleaves the initiator methionine (iMet) from Val1 of α-globin and βS-globin. Kinetic studies with MetAP2 show that βS-globin is a fivefold better substrate than α-globin. Knockdown of MetAP2 in human umbilical cord blood–derived erythroid progenitor 2 cells shows more extensive modification of α-globin than β-globin, consistent with kinetic data. Treatment of human erythroid cells in vitro or Townes SCD mice in vivo with selective MetAP2 inhibitors extensively modifies both globins with N-terminal iMet and acetylated iMet. HbS modification by MetAP2 inhibition increases oxygen affinity, as measured by decreased oxygen tension at which hemoglobin is 50% saturated. Acetyl-iMet modification on βS-globin delays HbS polymerization under hypoxia. MetAP2 inhibitor–treated Townes mice reach 50% total HbS modification, significantly increasing the affinity of RBCs for oxygen, increasing whole blood single-cell RBC oxygen saturation, and decreasing fractional flow velocity losses in blood rheology under decreased oxygen pressures. Crystal structures of modified HbS variants show stabilization of the nonpolymerizing high O2–affinity R2 state, explaining modified HbS antisickling activity. Further study of MetAP2 inhibition as a potential therapeutic target for SCD is warranted.

Introduction:Novel and safe therapeutic targets to increase expression of fetal hemoglobin (HbF) have potential to treat b-hemoglobinopathies (Platt, Brambilla et al. 1994, Steinberg 2020), including sickle cell disease (SCD) in which red blood cell (RBC) hemoglobin S resulting from a mutation in the hemoglobin β-globin subunit causes RBC sickling and hemolysis triggering vascular inflammation (Piel, Steinberg et al. 2017, Kato, Piel et al. 2018). Serum- and glucocorticoid-regulated kinase 1 (SGK1) is a serine/threonine kinase in the AGK kinase family that controls physiological processes such as cell growth, proliferation, migration, and apoptosis (Hayashi, Tapping et al. 2001, Sang, Kong et al. 2020). SGK1 is regulated by multiple ligands (insulin, cAMP, IGF-1, steroids, IL-2 and TGF-β) and phosphorylation by SGK1 modulates the activity of downstream effectors including ion channels (ENaC), Na-Cl cotransporters (NCC), membrane transporters, cellular enzymes (GSK3B) and transcription factors (FOXO3a, β-catenin, NF-κB and SP1) (Brunet, Park et al. 2001, Snyder, Olson et al. 2002, Loffing, Flores et al. 2006, Bruhn, Pearson et al. 2010, Boccitto and Kalb 2011, Wang, Hu et al. 2017). Previous studies show that SGK1 mediates survival signals in HEK cells by inhibiting FOXO3a through phosphorylation at Ser-315 (Brunet, Park et al. 2001). Recently, metformin was shown to induce HbF in erythroid cells through FOXO3a activation and metformin prevents RBC sickling in SCD (Zhang, Paikari et al. 2018). Thus, we hypothesized that inhibition of SGK1 and subsequent alleviation of SGK1-induced FOXO3a inhibition, may induce expression of erythroid cell HbF.

No relevant conflicts of interest to declare.

Leukotriene A4(LTA4) hydrolase catalyzes a rate-limiting final biosynthetic step of leukotriene B4(LTB4), a potent lipid chemotatic agent and proinflammatory mediator. LTB4has been implicated in the pathogenesis of various acute and chronic inflammatory diseases, and thus LTA4hydrolase is regarded as an attractive therapeutic target for anti-inflammation. To facilitate identification and optimization of LTA4hydrolase inhibitors, a specific and efficient assay to quantify LTB4is essential. This article describes the development of a novel 384-well homogeneous time-resolved fluorescence assay for LTB4(LTB4HTRF® assay) and its application to establish an HTRF-based LTA4hydrolase assay for lead optimization. This LTB4HTRF assay is based on competitive inhibition and was established by optimizing the reagent concentration, buffer composition, incubation time, and assay miniaturization. The optimized assay is sensitive, selective, and robust, with a Z´ factor of 0.89 and a subnanomolar detection limit for LTB4. By coupling this LTB4HTRF assay to the LTA4hydrolase reaction, an HTRF-based LTA4hydrolase assay was established and validated. Using a test set of 16 LTA4hydrolase inhibitors, a good correlation was found between the IC50values obtained using LTB4HTRF with those determined using the LTB4enzyme-linked immunoassay (R= 0.84). The HTRF-based LTA4hydrolase assay was shown to be an efficient and suitable assay for determining compound potency and library screening to guide the development of potent inhibitors of LTA4hydrolase.

A therapeutic antibody candidate (AT-19) isolated using multivalent phage display binds native tomoregulin (TR) as a mul-timer not as a monomer. This report raises the importance of screening and selecting phage antibodies on native antigen and reemphasizes the possibility that potentially valuable antibodies are discarded when a monomeric phage display system is used for screening. A detailed live cell panning selection and screening method to isolate multivalently active antibodies is described. AT-19 is a fully human antibody recognizing the cell surface protein TR, a proposed prostate cancer target for therapeutic antibody internalization. AT-19 was isolated from a multivalent single-chain variable fragment (scFv) antibody library rescued with hyperphage. The required multivalency for isolation of AT-19 is supported by fluorescence activated cell sorting data demonstrating binding of the multivalent AT-19 phage particles at high phage concentrations and failure of monovalent particles to bind. Pure monomeric scFv AT-19 does not bind native receptor on cells, whereas dimeric scFv or immunoglobulin G binds with nanomolar affinity. The isolation of AT-19 antibody with obligate bivalent binding activity to native TR is attributed to the use of a multivalent display of scFv on phage and the method for selecting and screening by alternate use of 2 recombinant cell lines.

A therapeutic antibody candidate (AT-19) isolated using multivalent phage display binds native tomoregulin (TR) as a multimer not as a monomer. This report raises the importance of screening and selecting phage antibodies on native antigen and reemphasizes the possibility that potentially valuable antibodies are discarded when a monomeric phage display system is used for screening. A detailed live cell panning selection and screening method to isolate multivalently active antibodies is described. AT-19 is a fully human antibody recognizing the cell surface protein TR, a proposed prostate cancer target for therapeutic antibody internalization. AT-19 was isolated from a multivalent single-chain variable fragment (scFv) antibody library rescued with hyperphage. The required multivalency for isolation of AT-19 is supported by fluorescence activated cell sorting data demonstrating binding of the multivalent AT-19 phage particles at high phage concentrations and failure of monovalent particles to bind. Pure monomeric scFv AT-19 does not bind native receptor on cells, whereas dimeric scFv or immunoglobulin G binds with nanomolar affinity. The isolation of AT-19 antibody with obligate bivalent binding activity to native TR is attributed to the use of a multivalent display of scFv on phage and the method for selecting and screening by alternate use of 2 recombinant cell lines.

Discoveries that lead to ZK 807834 (CI-1031, 2a), a potent and selective factor Xa (fXa) inhibitor currently in clinical testing as an intravenous antithrombotic, were initiated by the identification of the potent (Z,Z)-isomer of BABCH (1c). A structure-activity relationship (SAR) was established with a series of analogues of BABCH. This SAR database, combined with compu-ter modeling, demonstrated that binding of the second basic group in the S3 S4 pocket provided fXa potency and that a carboxylic acid group on the opposite side of the molecule resulted in selectivity versus thrombin. Simple substitution of a cyclic urea for the unsaturated ketone structure of BABCH gave disappointing results, but discovery of the bisphenoxy-pyridine analogues provided a template that could be readily optimized. The SAR established for this template is described and compared with computer modeling, REDOR NMR and X-ray crystallography studies. Inhibitor binding to fXa was increased by the introduction of a hydroxyl group on the proximal phenylamidine ring and by the introduction of fluorine atoms at C-3 and C-5 of the pyridine ring. Pharmacokinetic parameters were improved by balancing the contributions from the substituents on the distal ring and the central pyridine ring. The optimal combination was a methyl-(2H)-imidazoline group on the distal ring and a sarcosine at C-4 of the pyridine ring. The promising preclinical database for CI-1031 is described. This review relates the SAR leading to the discovery of the clinical candidate, CI-1031 directly to our best understanding of how this potent inhibitor interacts with the fXa active site.

Inhibition of factor Xa (FXa) may interrupt thrombus progression. This study compared the antithrombotic activity of a novel FXa inhibitor, ZK-807834 MW, 527 D; Ki(human FXa), 0.11 nM, with recombinant tick anticoagulant peptide rTAP; MW, 6,685 D; Ki, (human FXa) = 0.28 nM, and DX-9065a MW 445 D, Ki(human FXa), 40 nMin rabbits with arterial thrombosis induced by electrical vascular injury. ZK-807834 also was compared with low molecular weight heparin (LMWH; MW, 5,500 D) during venous thrombosis induced by placing a copper wire and threads in the vena cava. Inhibitors were administered as an i.v. bolus and 2-h infusion. Total dosages of ZK-807834, ≥0.7 μmol/kg (n = 18); rTAP, ≥1 μmol/kg (n = 18); or DX-9065a, ≥11 μmol/kg (n = 18) decreased the incidence of arterial thrombotic occlusion compared with control animals (p < 0.05). However, five of six animals given the lowest effective dosage of rTAP and four of six animals given DX-9065a bled from a surgical incision >5 min, but only two of six animals given ZK-807834 bled >5 min. Venous clot weights were reduced compared with controls for dosages of ZK-807834 ≥0.007 μmol/kg (n = 36) or LMWH ≥0.2 μmol/kg (n = 18). Prothrombin time (PT) and activated partial thromboplastin time (aPTT) were unchanged from baseline at the minimally effective dose of ZK-807834, whereas aPTT was increased twofold at the effective dose of LMWH. Thus ZK-807834 may be useful to attenuate thrombosis at lower dosages and with less perturbation of systemic hemostasis compared with available agents.

Factor Xa is a serine protease which activates thrombin (factor IIa) and plays a key regulatory role in the blood‐coagulation cascade. Factor Xa is, therefore, an important target for the design of anti‐thrombotics. Both factor Xa and thrombin share sequence and structural homology with trypsin. As part of a factor Xa inhibitor‐design program, a number of factor Xa inhibitors were crystallographically studied complexed to bovine trypsin. The structures of one diaryl benzimidazole, one diaryl carbazole and three diaryloxypyridines are described. All five compounds bind to trypsin in an extended conformation, with an amidinoaryl group in the S1 pocket and a second basic/hydrophobic moiety bound in the S4 pocket. These binding modes all bear a resemblance to the reported binding mode of DX‐9065a in bovine trypsin and human factor Xa.

Studies have been carried out to investigate aspects of the structure of thrombomodulin, an endothelial cell glycoprotein that binds thrombin and accelerates both the thrombin-dependent activation of protein C and the inhibition of antithrombin III. We have determined the shape of Solulin™, a soluble recombinant form of human thrombomodulin missing the transmembrane and cytoplasmic domains, by electron microscopy of preparations rotary-shadowed with tungsten. Solulin appears to be an elongated molecule about 20 nm long that has a large nodule at one end and a smaller nodule near the other end from which extends a thin strand. About half of the molecules form bipolar dimers apparently via interactions between these thin strands. Electron microscopy of complexes formed between Solulin and human α-thrombin revealed that a single thrombin molecule appears to bind to the smaller nodule of Solulin, suggesting that this region contains the epidermal growth factor-like domains 5 and 6. Epidermal growth factor-like domains 1-4 comprise the connector between the small and large nodule, which is the lectin-like domain; the thin strand at the other end of the molecule is the carbohydrate-rich region. With chondroitin sulfate-containing soluble thrombomodulin produced from either human melanoma cells Bowes or Chinese hamster ovary cells, a higher percentage of molecules bound thrombin and, in some cases, two thrombin molecules were attached to one soluble thrombomodulin in approximately the same region. These structural studies provide insight into the structure of thrombomodulin and its interactions with thrombin as well as aspects of the mechanisms of its actions.

The solution structure has been determined for a 19-residue peptide that is fully folded at room temperature. The sequence of this peptide is based on the C-loop, residues 371-389, of the fourth epidermal growth factor-like domain of thrombomodulin, a protein that acts as a cofactor for the thrombin activation of protein C. Despite its small size, the peptide forms a compact structure with almost no repeating secondary structure. The results indicate the structure is held together by hydrophobic interactions, which in turn stabilize the two β-turns in the structure. The first β-turn in the C-loop represents a conserved motif that is found in the published structures of five other epidermal growth factor-like proteins. The critical role of Phe376in the stabilization of the first β-turn is consistent with mutagenesis data with soluble thrombomodulin. The results also show that a small subdomain of a larger protein can fold independently, and therefore it could act as an initiation site for further folding.

Demers: Sanofi: Employment. Sturtevant:Sanofi: Employment. Guertin:Sanofi: Employment. Gupta:Sanofi: Employment. Desai:Sanofi: Employment. Vieira:Sanofi: Employment. Hicks:Sanofi: Employment. Ismail:Sanofi: Employment. Safo:Sanofi: Consultancy, Research Funding; Virginia Commonwealth University: Patents & Royalties. Wood:Sanofi: Consultancy, Research Funding. Higgins:Sanofi: Consultancy, Research Funding. Light:Sanofi: Employment.

Dilution of HbS with non-sickling hemoglobin or hemoglobin with increased oxygen affinity is clinically beneficial in sickle cell disease. Aldehydes, including 5-HMF, tucaresol or GBT440, modify the N-terminus of HbS by reversible covalent imine formation generating modified forms of HbS that resist polymerization under low oxygen concentrations. In contrast to reversible imine formation by aldehydes, we hypothesize that stable modification of HbS will result from N-terminal retention of the initiator methionine (iMet) and subsequent N-terminal acetylation of the iMet (acetyl-iMet).

Gupta: Sanofi: Employment. Lessard:Sanofi: Employment. Moore:Sanofi: Employment. Duan:Sanofi: Employment. Hicks:Sanofi: Employment. Light:Sanofi: Employment. Krishnamoorthy:Sanofi: Employment.

Nuclear factor (erythroid derived-2)-like 2 (NRF2), a basic leucine zipper transcription factor, is sequestered in the cell cytosol by Keap1, a kelch domain protein. Under steady state, this interaction results in ubiquitination and proteasomal degradation of the NRF2 protein. Modification of critical cysteine residues leads to conformational changes in KEAP1, resulting in nuclear translocation of newly synthesized NRF2 and modulation of downstream gene expression. NRF2 activation by compounds that modify KEAP1 such as dimethyl fumarate has been shown to induce γ-globin in erythroid cell systems. Moreover, NRF2 alleviates oxidative stress associated with SCD by activating antioxidant enzymes including catalase, glutathione peroxidase and superoxide dismutase (SOD), that scavenge free radicals (Chirico and Pialoux 2012, Belcher, Chen et al. 2017, Krishnamoorthy, Pace et al. 2017).

Vieira: Bioverativ a Sanofi Company: Employment. Hong:Bioverativ a Sanofi Company: Employment, Equity Ownership. Desai:Bioverativ a Sanofi Company: Employment, Equity Ownership. Safo:Bioverativ a Sanofi Company: Consultancy; Virginia Commonwealth University: Employment. Light:Bioverativ a Sanofi Company: Employment, Equity Ownership.

Sickle cell disease (SCD) is a genetic hemoglobinopathy driven largely by a single codon mutation of the β-globin gene resulting in polymerization of hemoglobin S (HbS). Anti-sickling approaches that involve increasing the oxygen affinity of HbS to treat SCD are under development and offer the potential to directly prevent HbS polymerization and its downstream pathophysiology. Two such compounds, 5-hydroxymethylfurfural (5HMF) and voxelotor (GBT440) have entered clinical trials for SCD with promising results and exert their therapeutic effects by modifying the N-terminus of HbS α-globin chains to form a reversible Schiff base. Formation of this N-terminal adduct stabilizes the oxygen-bound R-state (in the R2 conformation) that increases the oxygen affinity of the altered HbS and delaying the polymerization of HbS. In addition, genetic and small molecule therapies designed to increase fetal hemoglobin (HbF) expression hold great potential for the treatment of SCD. Increasing the percentage of HbF in RBCs significantly slows sickling kinetics without affecting oxygen delivery. Combination approaches of high-O2-Hb modification with HbF inducing therapies clinically could result in increased efficacy in the treatment of SCD, but the impact of hemoglobin modifiers on fetal hemoglobin has not been reported. Our present studies investigated the effects of 5HMF and voxelotor in HbF-rich umbilical cord blood derived RBCs.

Krishnamoorthy: Biogen: Employment, Equity Ownership, Other: share holder. Gupta:Biogen: Employment, Equity Ownership, Other: share holder. Hobbs:Biogen: Employment, Equity Ownership, Other: shareholder. Loh:Biogen: Employment, Equity Ownership, Other: share holder. Light:Biogen: Employment, Equity Ownership, Other: share holder. Peters:Biogen: Employment, Equity Ownership, Other: share holder. Sturtevant:Arietis: Employment. Pace:Augusta University: Employment; Biogen: Research Funding. Nakamura:Riken Institute: Employment. Lucas:Biogen: Employment, Equity Ownership, Other: share holder. Vieira:Biogen: Employment, Equity Ownership, Other: share holder.

Sickle Cell Disease (SCD) is caused by a point mutation in the beta- chain of hemoglobin, triggering a complex pathophysiology resulting in recurrent, painful vaso-occlusive events (VOCs) and chronic hemolytic anemia. Induction of fetal hemoglobin (HbF), is a well-established approach towards the treatment and potentially cure for SCD. This is exemplified by the condition known as Hereditary Persistence of Fetal Hemoglobin (HPFH), which when present in SCD patients results in asymptomatic disease. Reactivation of fetal hemoglobin can be achieved by different methodologies both genetic manipulation and pharmacological agents. The mechanism of action of hydroxyurea, the only FDA approved drug for the treatment of SCD, is by activating HbF. We have investigated the role of Nrf2 in activating fetal hemoglobin and it potential to ameliorate the symptoms of SCD. Nrf2 is a basic leucine zipper transcription factor that is bound to a cytosolic protein Keap1, a Kelch domain containing protein, which targets the factor to the proteasome. Previous studies have shown that chemical activation of Nrf2 leads to induction of HbF. Here we show that siRNA based knockdown of Keap1 led to ~70 % reduction in Keap1 mRNA levels in Human Umbilical cord Derived Eryrthroid Progenitor cells (HUDEP), and concomitantly mediated a 2-fold induction of g-globin mRNA levels. Peripheral blood derived mononuclear cells differentiated into erythroid progenitors treated with bardoxolone-Me, a potent Nrf2 activator, resulted in a significant 4-fold induction in γ-globin mRNA, 2.5 fold induction in F-cells and up to 2.5-fold up-regulation in both Aγ and Gγ globin protein. Bardoxolone-Me treated erythroid progenitors also demonstrated a significant reduction in hypoxia-induced shape changes of sickle cells - about 42 % decrease at 100 nM and 55 % decrease at 250 nM. Bardoxolone-Me was also evaluated in a Townes SCD mouse model carrying an inducible γ-globin gene. A single oral dose of bardoxolone-Me at 30mg/kg, showed 9-fold induction in γ-globin mRNA levels at 6 h compared to untreated mice. Based on these findings, Nrf2 activation, can potentially be utilized for HbF induction in the treatment of SCD.

van der Flier: Biogen: Employment. Liu:Biogen: Employment. Liu:Biogen: Employment, Equity Ownership, Honoraria, Research Funding. Mercury:Biogen: Employment. Ismail:Biogen: Employment. Seth-Chhabra:Biogen: Employment, Equity Ownership, Honoraria, Patents & Royalties, Research Funding. Kulman:Biogen: Employment. Schellenberger:Amunix Operating Inc: Employment. Light:Biogen: Employment, Equity Ownership. Peters:Biogen: Employment.

Krishnamoorthy: Biogen: Employment, Equity Ownership. Gupta:Biogen: Employment, Equity Ownership. Sturtevant:Biogen: Employment. Li:Georgia Regents University: Employment. Makala:Georgia Regents University: Employment. Hobbs:Biogen: Employment, Equity Ownership. Light:Biogen: Employment, Equity Ownership.

Sickle cell disease (SCD) is caused by a point mutation in the β-chain of hemoglobin, which triggers a complex pathophysiology resulting in recurrent, painful vaso-occlusive crises (VOC) and chronic hemolytic anemia. Fetal hemoglobin (HbF) is the major species of hemoglobin during fetal and neonatal development, the expression of which is replaced by the adult beta globin perinatally. Reactivation of HbF in adult SCD patients is considered beneficial in ameliorating the symptoms of the disease. This is exemplified by a condition known as hereditary persistence of fetal hemoglobin (HPFH) in heterozygous sickle disease patients where symptoms of SCD are absent and the typical HbF level is over 30% (Steinberg, M.H. et al, 2014). Strategies to reactivate HbF have been used successfully in mouse models of SCD and led to amelioration of the disease phenotype, which is also partly the mechanism of action of hydroxyurea, the only FDA approved drug for SCD. Up-regulation of HbF can be achieved by several different approaches including pharmacologic or genetic manipulation of transcription activators or repressors of HbF. Nuclear factor (erythroid derived-2)-like 2 (Nrf2) is a basic leucine zipper transcription factor that has been shown to activate γ-globin transcription and increase HbF levels in cultured CD34+ erythroid cells. Nrf2 is well established for its role in cytoprotective and anti-oxidant actions by transcriptionally activating target genes that confer protection from oxidative damage triggered from injury and inflammation. Nrf2 is normally sequestered in the cytoplasm by Keap1, a Kelch-domain protein. Release of Nrf2 from Keap1 allows nuclear translocation of Nrf2 and activation of target genes via its binding to an anti-oxidant response element (ARE) in the promoter region of Nrf2 target genes. Previous studies (Macari and Lowrey, 2011) have demonstrated that the γ-globin promoter contains an ARE sequence supporting Nrf2 as an inducer of HbF. We investigated the role of dimethyl fumarate (DMF), a small molecule Nrf2 agonist, in activating γ-globin transcription and enhancing levels of HbF in tissue culture and murine SCD models. Delayed-release DMF, approved by the FDA as Tecfidera, is an oral therapeutic for the treatment of relapsing multiple sclerosis (MS). After oral administration of DMF, human exposure occurs to both DMF and the bioactive primary metabolite, monomethyl fumarate (MMF). We assessed the ability of increasing concentrations of DMF to induce γ-globin mRNA in human erythroleukemia cells, CD34+ cells isolated from bone marrow of non-SCD volunteer donors, and peripheral blood mononuclear cells from SCD volunteer donors. γ-globin mRNA increased by 3-4 fold compared to control as quantitated using real-time PCR. DMF incubation also resulted in 2-fold upregulation in HbF protein levels as demonstrated by UPLC and western blotting analysis and also a 2-fold induction in percentage of RBC containing HbF (F-cells) by flow cytometry. In addition, co-incubation of DMF with hydroxyurea produced an additive effect on γ-globin mRNA (8 - 10 fold in non-SCD; 2-4 fold in SCD) and F-cell induction in CD34+ stem cell derived erythroid progenitors (3-4 fold in both SCD and non-SCD). DMF treatment of KU812 erythroleukemia cells induced Nrf2 activation resulting in Nrf2 nuclear translocation and occupancy of the ARE in the γ-globin promoter by ChIP assay. Moreover, siRNA based knockdown of Keap1 resulted in up-regulation of HbF by flow cytometry, demonstrating that activation of the Nrf2 pathway in erythroid cells can result in the up-regulation of HbF. The effect of DMF on HbF induction was assessed in the Townes SCD mouse model. Townes mice were administered DMF at 100 mg/kg IP for 5 day per week for one month duration and compared to treatment with vehicle control and to treatment with hydroxyurea. This dose and route of administration of DMF resulted in clinically relevant plasma levels of MMF in Townes mice. After a month of dosing, there was a 3- to 4-fold increase in circulating F-cells in the DMF treated mice which was comparable to the hydroxyurea treated mice. Based on these findings, DMF may have potential as an HbF inducer in the therapy of SCD, in addition to its vascular protective and heme detoxifying properties (Belcher et al, ASH 2014). Further clinical studies are needed to evaluate the safety and efficacy of DMF in SCD.

INTRODUCTION

Hines: Biogen Idec: Research Funding. Krishnamoorthy:Biogen Idec: Employment, Equity Ownership. White:Biogen Idec: Research Funding. Gupta:Biogen Idec: Employment, Equity Ownership. van der Flier:Biogen Idec: Employment, Equity Ownership. Peters:Biogen Idec: Employment, Equity Ownership. Jiang:Biogen Idec: Employment, Equity Ownership. Hobbs:Biogen Idec: Employment, Equity Ownership. Light:Biogen Idec: Employment, Equity Ownership.

Tan: Biogen Idec: Employment, Equity Ownership. Salas:Biogen Idec: Employment, Equity Ownership. Chen:Biogen Idec: Employment, Equity Ownership. Ashworth:Biogen Idec: Employment, Equity Ownership. Smith:Biogen Idec: Employment, Equity Ownership. Kistanova:Biogen Idec: Employment, Equity Ownership. Moore:Biogen Idec: Employment, Equity Ownership. Acosta:Biogen Idec: Employment, Equity Ownership. Kole:Biogen Idec: Employment, Equity Ownership. Light:Biogen Idec: Employment, Equity Ownership. Peters:Biogen Idec: Employment. Jiang:Biogen Idec: Employment, Equity Ownership.

Sickle cell disease (SCD) is caused by a point mutation in the beta-chain of hemoglobin, which triggers a complex pathophysiology resulting in recurrent, painful vaso-occlusive events (VOCs) and chronic hemolytic anemia. Among other abnormalities, sickle red blood cells (RBCs) are more adhesive than normal RBCs. Sickle RBC adhesion is an important pathway leading to both VOC and hemolysis: adhesive interactions promote the formation of blood flow-obstructing heterocellular aggregates that induce ischemic tissue damage and slow the transit of RBCs through the vasculature, promoting sickle hemoglobin polymerization and hemolysis. The capacity of sickle RBCs to adhere to endothelium positively correlates with disease severity. Interventions that reduce sickle RBC adhesion, in particular that of reticulocytes, by blocking specific molecular targets may limit or prevent disease sequelae. Very Late Antigen – 4 (VLA-4 or α4β1 integrin) is an important integrin on reticulocytes that mediates adhesive interactions to endothelial and plasma vascular cell adhesion molecule - 1 (VCAM-1), plasma fibrinogen, and other ligands. VLA-4 is expressed on the surface of sickle reticulocytes, with levels decreasing during maturation such that mature RBCs do not express surface VLA-4. Natalizumab is a recombinant humanized antibody that binds to the α4 subunit of VLA-4 and is used to treat multiple sclerosis (MS) and Crohn’s Disease (CD) by preventing leukocyte trafficking into tissues at sites of inflammation. We investigated the ability of natalizumab to block VLA-4 in the context of sickle whole blood, and evaluated the effect on sickle reticulocyte, mature erythrocyte, and leukocyte adhesion during physiologic flow conditions. Whole blood samples obtained from SCD donors were analyzed for saturation binding of natalizumab to surface VLA-4 on leukocytes and reticulocytes using flow cytometry. Up to 20% of reticulocytes from SCD donors (n=13) were positive for VLA-4 surface staining, whereas VLA-4 was undetectable on the surface of reticulocytes from healthy donors (n=4). VLA-4 on SCD reticulocytes and mononuclear leukocytes was saturated by natalizumab concentrations lower than known plasma trough concentrations achieved in MS and CD patients after natalizumab treatment, with SCD reticulocyte EC50= 0.11 ± 0.01 μg/mL and SCD leukocyte EC50= 0.16 ± 0.01 μg/mL (n=6). This translates to a binding affinity of 0.7 to 1.3 nM, similar to that found for healthy donor leukocytes. Both whole blood cells and isolated leukocytes from SCD donors adhered to immobilized VCAM-1 during physiologic flow conditions simulating post-capillary venules (shear stress =1 dynes/cm2) using a microfluidic flow-based adhesion system. Leukocytes from SCD donors adhered more to VCAM-1 (Mean = 20.7 + 10.0 from n=7) than leukocytes from healthy donors (Mean = 5.0 + 1.4 from n=5). The adhesion of leukocytes and reticulocytes to VCAM-1 was blocked by natalizumab in a dose-dependent manner and as a function of natalizumab saturation of cell surface VLA-4. Therapeutic IgG4 antibodies, including natalizumab, undergo chain shuffling in vivo with endogenous IgG4, leading to mono-specific IgG4 molecules, with one Fab arm specific to the antigen it was raised against. Compared to divalent natalizumab, monovalent chain-shuffled natalizumab bound to SCD reticulocytes at a7-fold higher EC50and inhibited SCD reticulocyte adhesion to VCAM-1 at higher antibody concentrations (10 and 1 µg/mL). While increased, these values are still below trough natalizumab levels achieved in natalizumab-treated patients. In summary, natalizumab bound VLA-4 on the surface of SCD reticulocytes and leukocytes similarly to healthy donor leukocytes and blocked adhesion of SCD reticulocytes and leukocytes to immobilized VCAM-1 under shear conditions. Natalizumab binding and adhesion inhibition occurred at plasma concentrations similar to those seen in natalizumab treated MS and CD patients. Based on these findings, natalizumab may have potential as an anti-adhesive therapy for SCD. Further clinical studies are needed to evaluate the safety and efficacy of natalizumab in SCD.

Recombinant activated factor VII (rFVIIa) is used for controlling bleeds in hemophilia A and B patients with inhibitory antibodies against factor VIII or factor IX. The hemostatic effect of rFVIIa at pharmacological doses is believed to be mediated by activating the tenase complex on platelets directly at the site of injury to promote thrombin generation. To increase the hemostatic activity of rFVIIa, we sought to target rFVIIa to platelets by fusing a single chain variable region of a monoclonal antibody against human platelet receptor αIIbβ3. The resulting fusion protein, FVII-189, displays higher affinity to human platelets and about 50-fold higher hemostatic activity than that of rFVIIa in whole blood from hemophilia A patients by rotational thromboelastometry (ROTEM). Here we investigated the safety, pharmacokinetics, and pharmacodynamics of FVII-189 in cynomolgus monkeys.

Tan: Biogen Idec: Employment, Equity Ownership. Chen:Biogen Idec: Employment, Equity Ownership. Salas:Biogen Idec: Employment, Equity Ownership. Peters:Biogen Idec: Employment, Equity Ownership. Light:Biogen Idec: Employment; Biogen Idec: Equity Ownership. Jiang:Biogen Idec: Employment; Biogen Idec: Equity Ownership.

rFVIIIFc is a fully recombinant fusion protein consisting of a single B domain-deleted human FVIII covalently attached to the dimeric Fc domain of human IgG1. rFVIIIFc has a 1.5-fold extended half-life and decreased clearance compared to rFVIII in patients with hemophilia A (Powell, Blood 2012). The Fc region of rFVIIIFc binds to the neonatal Fc receptor (FcRn), which is part of a naturally occurring pathway that cycles IgG back into circulation, delaying lysosomal degradation. Our previous studies with FcRn-chimeric mice showed that the decreased clearance of rFVIIIFc is mediated by FcRn expressed in somatic cells and not in hematopoietic cells. Biodistribution studies with 125I-rFVIIIFc identified liver as the major organ for rFVIIIFc disposition. Furthermore, qPCR studies showed that three different cell types in liver all express FcRn: hepatocytes (HC), liver sinusoidal endothelial cells (LSEC) and Kupffer cells (KC). In primary liver cell co-cultures, rFVIIIFc and rFVIII were both internalized by somatic LSEC and HC, but not KC, in the absence of von Willebrand factor (VWF). It has been reported that in vivo, 95% of FVIII circulates as a non-covalent complex with VWF (Lenting, JTH 2007) and we found that VWF delays liver uptake of FVIII and improves the circulating half-life of rFVIIIFc in mice.Compare the cellular localization of rFVIII and rFVIIIFc in the murine liver, in the presence and absence of VWF, in order to identify the cells responsible for the prolonged half-life of rFVIIIFc.FVIII KO (HemA) and FVIII/VWF DKO mice were dosed with rFVIII, rFVIIIFc or the mutants rFVIIIFc-IHH and rFVIIIFc-N297A that are deficient in interacting with FcRn and FcgR, respectively. The cellular localization in the liver of rFVIII, rFVIIIFc and mutants was investigated by immunohistochemistry, using a panel of anti-human FVIII monoclonal antibodies or anti-human-IgG (Fc) for detection, along with markers for LSEC, KC, and VWF.In HemA mice with circulating endogenous VWF, the majority of both rFVIII and rFVIIIFc signal is found in KC, which co-stain for VWF. In contrast, neither rFVIII nor rFVIIIFc is detected in the endothelial cells of the large vessels that stained for VWF in the Weibel-Palade bodies. Closer examination shows faint vesicular staining by rFVIII in HC in contrast to a diffuse staining by rFVIIIFc in the liver sinusoid. However in FVIII/VWF DKO mice lacking VWF, neither rFVIII nor rFVIIIFc is detected in KC consistent with the notion that internalization of FVIII by KC is mediated by VWF. The majority of rFVIII is found in HC, whereas rFVIIIFc again appears as a diffused liver sinusoidal staining pattern that is more intense than that observed in the HemA mice expressing VWF. These findings suggest free rFVIII is internalized and cleared by HC, while rFVIIIFc is cycled out of the HC and localizes in the Space of Disse, hence its sinusoidal localization. Alternately, in the absence of VWF, rFVIIIFc may cycle through LSEC rather than HC. In order to distinguish these two pathways, an rFVIIIFc variant, rFVIIIFc-IHH, which is not competent to bind FcRn, was tested. In a similar manner as rFVIII, the rFVIIIFc-IHH mutant localizes into HC in DKO mice and is found predominantly in KC in HemA with endogenous VWF. In contrast, rFVIIIFc-N297A, which is not competent to bind FcgR, localizes similarly to rFVIIIFc in DKO mice, excluding a role for FcgR.Our current immunohistochemical studies and previous biodistribution and PK studies in mice indicate that there are two parallel, linked clearance pathways for rFVIII and rFVIIIFc. rFVIII or rFVIIIFc complexed with endogenous VWF is internalized predominantly by macrophages, including KC. However, because the VWF-FVIII complex is in constant equilibrium, a fraction of free rFVIII or rFVIIIFc is available for clearance by HC. We propose that this free fraction of rFVIIIFc entering HC is then cycled by FcRn back into the liver sinusoid and into circulation, in contrast to the free rFVIII entering the HC. Staining of the rFVIIIFc-IHH mutant in HC suggests that these cells play a dominant role in vivo, however LSEC may also contribute to cycling of VWF-free rFVIIIFc. The fate of the VWF-bound rFVIIIFc fraction internalized by KC is less clear, however data using FcRn-chimeric mice suggest that FcRn expressed in hematopoietic cells, including KC contributes only marginally to the delayed clearance of rFVIIIFc.van der Flier: Biogen Idec: Employment, Equity Ownership. Liu:Biogen Idec: Employment, Equity Ownership. Light:Biogen Idec: Employment, Equity Ownership. Jiang:Biogen Idec: Employment, Equity Ownership.

Treatment of bleeding episodes in hemophilia patients with inhibitory antibodies to factor VIII or factor IX by recombinant activated factor VII (rFVIIa, NovoSeven) has been sub-optimal partly due to its low affinity to activated platelets and its short half-life in circulation. To develop a rFVIIa variant with enhanced coagulant activity, we have targeted rFVIIa to platelets by monoclonal antibodies that recognize the human platelet receptor αIIbβ3. However, the assessment of pharmacokinetic parameters in mice is limited by the lack of recognition of mouse αIIbβ3 by antibodies to human αIIbβ3. The present study addresses the need to develop appropriate in vivo models to study this new class of bypass therapeutics.

rFVIIIFc is a fully recombinant fusion protein consisting of a single B domain-deleted human FVIII covalently attached to the dimeric Fc domain of human IgG1. rFVIIIFc has a 1.5-fold extended half-life and decreased clearance compared to rFVIII in patients with hemophilia A (Powell, Blood 2012). The Fc region of rFVIIIFc binds to the neonatal Fc receptor (FcRn), which is part of a naturally occurring pathway that cycles IgG back into circulation, delaying lysosomal degradation. Our previous studies with FcRn-chimeric mice showed that the decreased clearance of rFVIIIFc is mediated by FcRn expressed in somatic cells and not in hematopoietic cells. Biodistribution studies with 125I-rFVIIIFc identified liver as the major organ for rFVIIIFc disposition. Furthermore, qPCR studies showed that three different cell types in liver all express FcRn: hepatocytes (HC), liver sinusoidal endothelial cells (LSEC) and Kupffer cells (KC). In primary liver cell co-cultures, rFVIIIFc and rFVIII were both internalized by somatic LSEC and HC, but not KC, in the absence of von Willebrand factor (VWF). It has been reported that in vivo, 95% of FVIII circulates as a non-covalent complex with VWF (Lenting, JTH 2007) and we found that VWF delays liver uptake of FVIII and improves the circulating half-life of rFVIIIFc in mice.

Recombinant fusion of a single molecule of either FVIII or FIX to the Fc domain of IgG1 (rFVIIIFc or rFIXFc, respectively) has been shown to decrease clearance compared to rFIX or rFVIII in an FcRn-dependent manner (Blood (2010) 115: 2057; Blood (2012) 119:3024). The long-lasting procoagulant activity of these two molecules relies on a natural pathway in somatic (endothelial) and hematopoietic (monocyte) cells that recirculate antibodies into the blood stream. In hemophilia A subjects, rFVIIIFc has demonstrated ∼1.6-fold longer half-life compared to recombinant full length FVIII (Advate®) (Blood (2012) 119:3031), while rFIXFc shows a ∼3-fold longer half-life than that reported for current rFIX products (Blood (2012) 119:666). In the current study we determined the biodistribution of rFVIIIFc in hemophilia A (HemA) mice and sought to identify the cell types and tissues that contribute to FcRn-mediated protection and recycling of rFVIIIFc or rFIXFc.The pharmacokinetics of rFVIIIFc or rFIXFc were compared to IgG1 in bone marrow chimeric mice of wild-type and FcRn-KO mice. The plasma FVIII activity was determined using a chromogenic assay, and the decay of antigen levels of rFIXFc or IgG1 in plasma post dosing was determined by ELISA. The biodistribution of iodinated rFVIIIFc in HemA mice was evaluated by Quantitative Whole Body Autoradiography (QWBA).We have shown that the rFVIIIFc half-life is ∼2-fold longer in wild type mice than in FcRn-KO mice. However, bone marrow transplantation of FcRn-expressing hematopoietic cells into FcRn-deficient mice did not substantially increase the half-life of rFVIIIFc. Also, bone marrow transplantation of FcRn-deficient cells into wild-type mice did not decrease the half-life of rFVIIIFc, suggesting a substantional role of somatic FcRn. In contrast, while rFIXFc has a ∼3 fold longer half-life in FcRn wild-type mice than in knockout mice, both FcRn-deficient mice receiving wild-type bone marrow, as well as wild-type mice receiving FcRn-deficient bone marrow, displayed clearance levels of rFIXFc intermediate to that of wild-type and FcRn-KO mice. These studies suggest that rFVIIIFc is recycled via FcRn predominantly in somatic cells, while rFIXFc, similar to IgG1, is recycled approximately equally in both FcRn-expressing hematopoietic and somatic cells.Furthermore, QWBA using 125I-labelled rFVIIIFc in HemA mice show extensive localization of rFVIIIFc in the liver, suggesting that somatic cells in the liver may mediate rFVIIIFc recycling through Fc – FcRn interaction. In summary, these data suggest that similar to IgG1, rFVIIIFc and rFIXFc are recycled in both somatic and hematopoietic FcRn-expressing cells. However, the relative contributions of the various cell types are dependent on the particular Fc-fusion protein, suggesting additional regulation at the level of protein-specific interactions for cellular uptake. Characterization of the cell-type specific recycling pathways for rFVIIIFc and rFIXFc is in progress to support further delineation of the mechanism of action for these two novel therapeutics currently in registrational trials for treatment of severe Hemophilia A and B, respectively.van der Flier: BiogenIdec: Employment. Liu:BiogenIdec: Employment. Drager:BiogenIdec: Employment. Vallee:former BiogenIdec: Employment. Chen:BiogenIdec: Employment. Tan:BiogenIdec: Employment. Wharton:former BiogenIdec: Employment. Light:biogenidec: Employment. Jiang:biogenidec: Employment.

Kulman: Biogen Idec: Employment. Bou-Assaf:Biogen Idec: Employment. Berkowitz:Biogen Idec: Employment. Light:biogenidec: Employment. Mezo:Biogen Idec: Employment. Wang:Biogen Idec: Employment. Whitlow:Colabrativ Inc.: Employment; Biogen Idec: Consultancy. Culyba:Biogen Idec: Employment. Peters:Biogen Idec: Employment.

Tan: BiogenIdec: Employment. Chen:BiogenIdec: Employment. van der Flier:BiogenIdec: Employment. Liu:BiogenIdec: Employment. Light:biogenidec: Employment. Jiang:biogenidec: Employment.

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