ALBERT

Description: Key Points The fraction of invariant NKT cells demonstrating activation is increased during painful crises compared with steady state. Regadenoson, an adenosine A2A receptor agonist, decreases the fraction of activated invariant NKT cells during painful crises.

Description: ADAMTS13 proteolysis of von Willebrand Factor (VWF) generates smaller multimers that are less likely to promote blood clotting. Deficiency of ADAMTS13 leads to thrombotic thrombocytopenic purpura, a frequently fatal disease, characterized by microangiopathic hemolytic anemia and thrombocytopenia. ADAMTS13 has a characteristic domain structure that includes metalloprotease and disintegrin domains, a thrombospondin type 1 repeat (TSR), cysteine-rich and spacer domains, 7 additional TSRs, and 2 carboxyl-terminal CUB domains. The ADAMTS13 substrate, VWF, is synthesized in endothelial cells and forms large multimers within the cell. These large multimers are secreted and adhere to the endothelial cell surface where they can bind platelets flowing in blood leading to thrombosis. ADAMTS13 has been shown to cleave VWF on the surface of endothelial cells, but it is unclear if ADAMTS13 also interacts with the endothelial cell surface. We have used iodinated ADAMTS13, fluorescence-activated cell sorting (FACS), and biochemical analysis using flow conditions to demonstrate that ADAMTS13 does interact with the endothelial cell surface. Iodinated ADAMTS13 bound the endothelial cell surface at 4oC. This binding was specific since the binding was inhibited in the presence of 40-fold excess unlabeled ADAMTS13. Binding of ADAMTS13 to the cell surface was time-dependent with maximal binding occurring within two hours. The binding was also reversible; the half-time for dissociation was four hours. Binding was inhibited by heparin but not by dextran sulfate. The Kd of binding to endothelial cells was 75 nM (range 40–100 nM). FACS analysis also demonstrated binding of ADAMTS13 to endothelial cells. A fluorescein isothiocyanate labeled anti-epitope antibody bound to endothelial cells in the presence but not the absence of ADAMTS13. A polyclonal antibody to VWF inhibited binding of ADAMTS13 to VWF, but this antibody did not affect binding of ADAMTS13 to endothelial cells, suggesting that ADAMTS13 interacts with endothelial cells independently of VWF. Studies with C-terminal truncation constructs of ADAMTS13 indicated that the carboxyl-terminal TSRs are important for binding since constructs terminating with the metalloprotease domain, the first TSR, or the sixth TSR failed to compete with full-length ADAMTS13 for binding to endothelial cells, but constructs terminating with either the seventh or eighth TSR did compete for binding. Lastly, recombinant ADAMTS13 was found to be associated with endothelial cells in flow experiments. Endothelial cells were perfused with medium containing plasma concentrations of ADAMTS13 (1 μg/ml) at 10 dynes/cm2. After perfusion, the endothelial cells were washed and bound ADAMTS13 was identified from whole cell lysates through SDS-PAGE and immunoblotting with an anti-V5 epitope antibody. ADAMTS13 was found associated with endothelial cells after perfusion. Binding of ADAMTS13 to the endothelial cells prior to perfusion led to enhanced proteolysis of VWF as compared to addition of ADAMTS13 during perfusion only. This suggests that the interaction of ADAMTS13 with endothelial cells is important since it enhances the cleavage of VWF as compared to that of ADAMTS13 in solution.

Description: VWF multimers are cleaved into smaller less thrombogenic fragments in plasma by ADAMTS13. Deficiency of ADAMTS13 activity leads to thombotic thrombocytopenic purpura (TTP), a disease characterized by thrombocytopenia, microangiopathic hemolytic anemia, fever, neurological decline, and renal insufficiency. ADAMTS13 is a member of the A Disintegrin and Metalloprotease with ThromboSpondin repeats family that has characteristic motifs including a signal sequence, a propeptide, a catalytic metalloprotease domain, a disintegrin domain, a thrombospondin-1 (TSP1) repeat, a cysteine-rich region, and a spacer domain. These domains are followed in ADAMTS13 by seven additional TSP1 repeats and two C-terminal CUB domains. Previous work has shown that ADAMTS13 truncated after the spacer domain retains proteolytic activity towards VWF, but little is known about the potential role of the additional C-terminal TSP1 and CUB domains in VWF binding or cleavage. We therefore developed an enzyme-linked immunosorbent assay (ELISA)-based system to study ADAMTS13-VWF binding. VWF was immobilized in microtiter wells and incubated with plasma or recombinant ADAMTS13 variants. Bound ADAMTS13 was detected directly by solubilization and Western blotting, or indirectly by ELISA. ADAMTS13 proteolytic activity towards VWF is enhanced by denaturation of VWF with urea or guanidine, but ADAMTS13 bound specifically to VWF without prior denaturation. EDTA increased the binding of ADAMTS13 to VWF and prevented proteolysis of the immobilized VWF. Binding was saturable and time-dependent with maximal binding in two hours. Binding was reversible with a half-time for dissociation of four hours. ADAMTS13 in normal human plasma but not in plasma from a patient with TTP bound immobilized VWF. The stoichiometry of VWF monomers to ADAMTS13 at saturation was approximately 2 (range 1–4) and the Kd of recombinant ADAMTS13 binding to VWF was 12 nM (range 5–26 nM). This Kd for binding is similar to the Km for VWF cleavage of 16 nM determined independently, and both are comparable to the estimated plasma ADAMTS13 concentration of 8 nM. The properties of C-terminally truncated ADAMTS13 constructs suggest a regulatory function for certain domains. Truncation after the 8th TSP1 domain decreased the Kd 2-fold. Further truncation after either the 6th or 7th TSP1 domain increased the Kd 2-fold relative to full-length ADAMTS13. Truncation after the spacer domain gave binding properties indistinguishable from full-length ADAMTS13. Truncation after the metalloprotease domain gave no detectable binding to VWF. Therefore, binding of ADAMTS13 to VWF requires sequences in the cysteine-rich or spacer domains, and is modulated by sequences in at least the 8th TSP1 repeat and the CUB domains.

Description: Patients with sickle cell disease have an increased number of circulating activated iNKT cells while murine SCD models report increased number and activation state of iNKT cells in target organs. Furthermore, the use of a murine iNKT cell-depleting antibody in murine SCD models prevents inflammation driven end-organ damage. NKTT120 is a humanized monoclonal antibody directed to the unique invariant TCR of iNKT cells that depletes these cells by ADCC. In preclinical studies, NKTT120 has demonstrated a safe and specific dose and time dependent depletion/recovery of iNKT cells. The preclinical efficacy and safety data supported a clinical development program to show that NKTT120 demonstrates the same safety and specificity for iNKT cell depletion from the peripheral circulation in SCD patients. In this first in human phase 1 dose-escalation study, we have examined the safety of NKTT120 in adults with steady state SCD. Future studies will explore the ability of NKTT120 prevent painful vaso-occlusive crises. Objective: To determine the safety, maximum tolerated dose (MTD), pharmacokinetics, and pharmacodynamics of NKTT120 in adults with steady state SCD. The optimal dose for a phase 2 study of NKTT120 will deplete iNKT for approximately 3 months allowing for periodic dosing. Methods: A first-in-humanphase 1 study utilizing a 3+3 design to evaluate single doses escalating over a range of 7 doses from 0.001 mg/kg to 1.0 mg/kg. The primary outcome measure is safety. Secondary outcomes include blood iNKT cell depletion and recovery, pain, analgesic use, quality of life (QoL), and pulmonary function. During a screening run-in period and after dosing of NKTT120, subjects maintained a daily smartphone eDiary (eSCaPe) to report pain, respiratory symptoms and analgesic use. ASCQ-Me and PROMIS QoL questionnaires were administered at clinic visits. The screening run-in outcomes will be used as baseline comparison for values obtained post-dosing. Results: A total of 21 patients were enrolled into the 7 cohorts of the completed and closed study. The drug was delivered as a 10-minute IV push in all cohorts. No MTD was defined, as no DLTs were reported. Three subjects each were dosed at 0.001, 0.003, 0.01, 0.03, 0.1, 0.3 or 1.0 mg/kg. At leastone month of follow-up data on circulating iNKT cell numbers are available for all of the patients dosed in the study. Only iNKT cell counts were affected by NKTT120 dosing, no change in other hematologic parameters was observed in peripheral blood. No acute elevation in circulating inflammatory cytokines was seen after antibody administration. All doses of NKTT120 resulted in maximum depletion of iNKT cells at the first time point (6 hours) monitored in all patients. During the recovery period, all patients had detectable iNKT cells in their peripheral blood. In all cohorts, the time to recovery of iNKT cells correlates with the starting circulating levels, with a longer recovery time for patients with lower baseline cell numbers. T1/2 is approximately 11 days. As observed in the pre-clinical safety studies, iNKT cell depletion and recovery was dose and time dependent. At the recommended Phase 2 dose (0.3 mg/kg) no iNKT cells were detectable in the peripheral circulation for a period of several months, suggesting near complete tissue depletion at these doses requiring recovery from T cell precursors that are not targeted by NKTT120. Conclusions: In adults with SCD,NKTT120 administered up to a dose of 1.0 mg/kg specifically reduces iNKT cells without NKTT120 dose limiting toxicity. Patients at the higher dose cohorts of NKTT120 illustrate temporal pattern for iNKT cell depletion and recovery in the circulation that inform the dosing strategy for phase 2 studies. The recommended Phase 2 dose is 0.3 mg/kg administered at a 3 month interval. The Phase 2 study will highlight the reduction of iNKT cells in the suppression of the inflammatory stimuli that promote many of the pathophysiologic sequelae seen in SCD. Disclosures Eaton: NKT Therapeutics: Employment. Mazanet:NKT Therapeutics: Consultancy, Equity Ownership. Nathan:NKT Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Description: ADAMTS13 is a plasma metalloproteinase that limits platelet-rich thrombi by cleaving von Willebrand factor (VWF) at the Tyr1605-Met1606 bond in the A2 domain. A minimal substrate that consists of GST linked to VWF residues Asp1596-Arg1668 with a C-terminal 6×His tag (GST-VWF73) is cleaved rapidly by plasma ADAMTS13. Further removal of Glu1660-Arg1668, which comprises a predicted C-terminal α-helix (GST-VWF64) markedly reduced the rate of cleavage, suggesting this helix comprises an exosite for substrate recognition. By amino acid sequencing, ADAMTS13 was shown to cleave the Tyr1605-Met1606 bond of GST-VWF64. Truncation of ADAMTS13 after certain structural domains had different effects on substrate cleavage. After removal of the GST moiety by proteolysis, the kinetic constants for cleavage of VWF73 and VWF64 were determined for several recombinant ADAMTS13 variants using a quantitative MALDI-MS assay (Table). Comparison of the specificity constants (kcat/Km) shows that ADAMTS13 truncated after the spacer domain (construct MDTCS) cleaved VWF73 ~20-fold faster than did a similar enzyme without the spacer domain (construct MDTC). In contrast, both MDTCS and MDTC cleaved VWF64 slowly at a rate similar to the cleavage of VWF73 by MDTC. Most of the variation in cleavage rates was explained by differences in Km, suggesting that the spacer domain recognizes an exosite at the C-terminus of VWF73 that is missing from VWF64. Cleavage of VWF73 yields a C-terminal product (cVWF63). Purified cVWF63 (7.5 μM) inhibited MDTCS activity toward VWF73 or VWF64 (1.5 μM) by »90%, but did not inhibit MDTC, suggesting that the exosite of VWF73 or cVWF63 interacts directly with the spacer domain. Moreover, cVWF63 inhibited the cleavage of multimeric VWF by full-length ADAMTS13 and MDTCS up to 80%, but did not inhibit MDTC. The selective effects of deleting the ADAMTS13 spacer domain and Glu1660-Arg1668 of the VWF domain A2 suggest that the C-terminal exosite of the VWF A2 domain accelerates substrate cleavage by binding specifically to the ADAMTS13 spacer domain. Table Kinetics studies of VWF73 and VWF64 cleavage by ADAMTS13 and its truncations VWF73 VWF64 Enzyme K m (μM) k cat (s−1) k cat /K m (×105 M−1 s−1) K m (μM) k cat (s−1) k cat /K m (×105 M−1 s−1) FL-ADAMTS13 1.7±0.4 1.3±0.1 7.5±2.0 37.7±12.8 1.9±0.4 0.5±0.3 MDTCS 0.8±0.2 1.7±0.1 20.5±6.6 5.5±1.4 0.6±0.1 1.0±0.3 MDTC 16.0±4.5 1.8±0.3 1.1±0.5 17.9±6.3 1.6±0.3 0.9±0.5

Description: Background: von Willebrand disease (VWD) is the most common congenital bleeding disorder. In affected women, menorrhagia is the most common bleeding symptom. Combined oral contraceptives (COCs), the first choice therapy recommended by NHLBI 2007 guidelines, reduce menstrual loss by increasing coagulation factor levels, but at least 30% are unresponsive or intolerant. Non-hormonal options include the antifibrinolytic tranexamic acid (TA), reduces menstrual bleeding by 30-50%, but requires dosing three times a day. Intranasal desmopressin (Stimate) is simpler to use, but ineffective in ~20%. Effective treatment for menorrhagia, thus, remains the greatest unmet health need in women with VWD. Von Willebrand factor (VWF) is used typically when first-line and second-line treatments fail, but few data exist regarding its effectiveness in reducing menorrhagia. Methods: We therefore conducted a survey of U.S. hemophilia treatment centers (HTCs) of current therapy for menorrhagia in VWD, utilizing Centers for Disease Control (CDC) website https://www2a.cdc.gov/ncbddd/htcweb/Main.asp, and the Hemostasis and Research Society (HTRS) site http://htrs.org. To specifically assess the use of VWF concentrate for menorrhagia, we also performed a literature review using medical subject heading (MeSH) search terms "von Willebrand factor," "menorrhagia," and "von Willebrand disease." Statistical analysis was by descriptive statistics. Results: Of 83 surveys distributed to hemophilia treatment centers (HTCs) caring for adult patients, 35 HTC MDs responded (42.2%) but only 20 HTC MDs (24.1%) provided sufficient data for analysis. These 20 HTC MDs reported a total of 1,321 women with VWD age 18-45 years seen during the 3-year period 2011-2014, of whom 816 (61.8%) had menorrhagia. Among these women, the most common first-line therapy was COCs, reported by 50.0% of the 20 HTC MDs, TA in 30.0% and desmopressin (DDAVP) in 20.0%. Overall, including all therapies (first-, second-, third-line), DDAVP was prescribed by 90.0% of the 20 HTC MDs, TA in 80.0%, COCs in 70.0%, aminocaproic acid (amicar) in 25.0%, and the levonorgestrel-releasing intrauterine system (Mirena IUD) in 15.0%. Only 4 HTC MDs (20.0%) prescribed VWF concentrate (VWF) for menorrhagia: all used VWF as third-line therapy after first-line and second-line treatments had failed. In the 13 women with type 1, 2, or 3 VWD and menorrhagia treated with intravenous VWF by these 4 HTC MDs, there was reduction in menorrhagia in all 13 (100%), with no adverse effects. These patients learned intravenous technique and infused VWF at 40-50 IU/kg at home for up to 5 days of menstrual bleeding each cycle, with good acceptability. In the literature search, we identified six published studies, including two prospective clinical trials, two retrospective observational trials, and two observational network studies. A total of 455 subjects with VWD reported in these six studies were treated with either plasma-derived (pdVWF) or recombinant (rVWF) VWF for bleeds. Of these, nearly one-third or 138 (30.3%) were women with type 1, 2, or 3 VWD and menorrhagia who were treated with pdVWF or rVWF at a dose of 36-50 IU/kg for 1-6 days of menstrual cycle bleeding. In these studies, 95-100% of these women reported reduction in menorrhagia, with no reported adverse events. Discussion: This survey and literature review of 151 women with VWD and menorrhagia represent the largest treatment experience to date. DDAVP, TA, and COCs are the most common first-line therapies. VWF is a third-line therapy but safely and effectively reduces menorrhagia in at least 95% of women with VWD. Prospective clinical trials of VWF are needed to establish the minimal dose required for menorrhagia, to determine patient acceptability of this intravenous therapy, and to compare safety and efficacy with standard therapy. Disclosures Off Label Use: recombinant VWF and plasma-derived VWF for treatment of menorrhagia in VWD. Ragni:Bristol Myers Squibb: Research Funding; Biogen: Research Funding; Bayer: Research Funding; Baxalta: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Alnylam: Research Funding; Dimension Therapeutics: Research Funding; CSL Behring: Research Funding; Foundation Women Girls Blood Disorders: Membership on an entity's Board of Directors or advisory committees; Genentech Roche: Research Funding; Medscape, Web MD: Honoraria; National Hemophilia Foundation: Membership on an entity's Board of Directors or advisory committees; Pfizer: Research Funding; SPARK: Research Funding; Shire: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tacere Benitec: Membership on an entity's Board of Directors or advisory committees; Ferring Pharmceuticals: Research Funding; Biomarin: Research Funding; Vascular Medicine Institute: Research Funding. Malec:Baxalta: Research Funding; Biogen: Research Funding. Coyle:Bayer: Membership on an entity's Board of Directors or advisory committees. Drygalski:Biogen: Consultancy; Baxalta: Consultancy; Novo Nordisk: Consultancy; Pfizer: Consultancy; Bayer: Consultancy; Hematherix Inc: Equity Ownership; Biogen: Research Funding; Baxalta: Research Funding; Novo Nordisk: Research Funding; Bayer: Research Funding; CSL Behring: Speakers Bureau; Hematherix Inc: Membership on an entity's Board of Directors or advisory committees. James:CSL Behring: Membership on an entity's Board of Directors or advisory committees; Baxter: Membership on an entity's Board of Directors or advisory committees. Jobe:Biogen: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; CSL-Behring: Membership on an entity's Board of Directors or advisory committees. Konkle:Octapharma: Research Funding; Baxalta: Consultancy; CSL Behring: Consultancy; Baxalta: Research Funding. Kouides:CSL Behring: Membership on an entity's Board of Directors or advisory committees. Kuriakose:Kedrion: Speakers Bureau. Ma:Baxalta: Membership on an entity's Board of Directors or advisory committees, Research Funding; Biogen Idec: Membership on an entity's Board of Directors or advisory committees, Research Funding; Kedrion: Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees. Nance:Patient Services, Inc.: Membership on an entity's Board of Directors or advisory committees. Neff:Alexion: Membership on an entity's Board of Directors or advisory committees; Novonordisk: Research Funding; Baxter: Membership on an entity's Board of Directors or advisory committees; Kedrion: Membership on an entity's Board of Directors or advisory committees; Novonordisk: Membership on an entity's Board of Directors or advisory committees. Philipp:Baxter: Research Funding. Yaish:Agios: Membership on an entity's Board of Directors or advisory committees.

Description: An increased number of circulating activated iNKT cells have been reported in sickle cell disease patients, and in mouse studies, iNKT cells have been reported to have increased number and activation state in target organs. Depletion of iNKT cells in a mouse model of sickle cell disease decreases inflammation and prevents end-organ damage. NKTT 120 is a humanized monoclonal antibody directed against the iNKT cell invariant TCR that depletes these cells by antibody dependent cellular cytotoxicity. Preclinical studies show that NKTT120 has high affinity and specificity for iNKT cells. NKTT120 administration as a 10 minute IV infusion produces depletion and recovery of iNKT cells in a dose and time dependent manner. Our global hypothesis is that NKTT120 will deplete iNKT cells, reduce inflammation and prevent painful vaso-occlusive crises. In this phase 1 dose-escalation study, we have examined the safety of NKTT120 in adults with steady state SCD. Objective: To determine the safety, maximum tolerated dose (MTD), pharmacokinetics, and pharmacodynamics of NKTT120 in adults with steady state SCD. The optimal dose for a phase 2 study of NKTT120 will deplete iNKT for approximately 3 months allowing for periodic dosing. Methods: Phase 1 study utilizing a 3+3 design to evaluate single doses escalating over a range of 5 doses from 0.001 mg/kg to 0.3 mg/kg thus far. The primary outcome measure is safety. Secondary outcomes include blood iNKT cell number depletion and recovery, pain, analgesic use, quality of life (QoL), and pulmonary function. During a screening run-in period and after dosing of NKTT120, subjects maintained a daily smartphone eDiary (eSCaPe) to report pain, respiratory symptoms and analgesic use. ASCQ-Me and PROMIS QoL questionnaires were administered at clinic visits. The screening run-in outcomes will be used as baseline comparison for values obtained post-dosing. Results: At leastone month of follow-up data on iNKT cell numbers are available for the first eighteen patients dosed in the study. Three subjects were dosed at each dose level of 0.001, 0.003, 0.01, 0.03, and 0.1 and 0.3 mg/kg. Only iNKT cell counts were affected by NKTT120 dosing. No change in other hematologic parameters was observed. Eleven of 15 subjects in the first 5 cohorts had recovered iNKT cells by 28 days. Of the remaining 4 patients, 3 recovered iNKT cells by 56 days and the last recovered iNKT cells within 5 months. No patient has recovered iNKT cells in cohort 6 (0.3 mg/kg) within 28 days. Time to recovery of iNKT cells correlates with the starting circulating levels, with a longer recovery time for lower cell number. NKTT120 has been well tolerated with no dose limiting toxicities reported. Conclusions: In steady state adults with SCD,NKTT120 administered up to a dose of 0.3 mg/kg specifically reduces iNKT cells without NKTT120 dose limiting toxicity. Patient cohorts at higher doses of NKTT120 are planned to further define the most effective dose and dose interval for iNKT cell depletion and recovery in the blood and target tissues. The final dose selection will support longer term studies on the reduction of iNKT cells in the suppression of the inflammatory stimuli that promote many of the pathophysiologic sequelae seen in SCD. Disclosures Vichinsky: ApoPharma: Research Funding; ARUP Research labs: Research Funding. Eaton:NKT Therapeutics: Employment. Mazanet:NKT Therapeutics: Employment.

Description: Abstract 849FN2 Adenosine 2A receptor (A2AR) agonists decrease pulmonary inflammation and injury in a murine model of sickle cell disease (SCD) by interrupting invariant NKT (iNKT) cell activation that occurs as a result of vaso-occlusion (Clin Immunol 2011). iNKT cells represent a subpopulation of T lymphocytes that rapidly generates pro-inflammatory cytokines upon activation and thereby may play a role in sustaining or propagating vaso-occlusion in human SCD. To evaluate the effects of A2AR activation in human SCD, we are conducting a dose-seeking and safety study of the FDA-approved, highly selective A2AR agonist regadenoson in patients with SCD ≥ 14 years of age. This is a multi-center, phase I clinical trial of infusional regadenoson that utilizes a 3+3 design and is comprised of four stages to determine maximum tolerated dose and safety during a 12 hour infusion in adults at baseline (defined as absence of increased pain, ED or hospital visit in the past 2 weeks) (stage 1), a 24 hour infusion in adults at baseline (stage 2), a 24 hour infusion in adults during a painful vaso-occlusive crisis (VOC) (stage 3), and a 24 hour infusion in children (≥ 14 years) during a painful VOC (stage 4). Three dose levels of infusional regadenoson have been evaluated thus far: 0.24 (level 0), 0.6 (level 1) and 1.44 mcg/kg/hour (level 2). Samples for plasma levels and iNKT cell activation are obtained at 0, 6, 12, 18 and 30 hours after start of infusion. Two methods are used to identify iNKT cells: 1) CD1d tetramers, loaded with the α-GalCer-like glycolipid PBS57 (defined as tetramer+CD3+), and 2) 6B11 antibody to the invariant T cell receptor (defined as CD45+6B11 high). NF-kB activation is measured using phospho-Ser536-p65 antibody (phospho-NF-kB). To date, 15 subjects have received infusional regadenoson. Regadenoson appears to be safe (devoid of cardiovascular or other side effects) and biologically active in reducing iNKT cell activation markers during a 12 hour infusion at dose levels 1 and 2, and we are currently evaluating the safety of a 24 hour infusion at dose level 2. No dose limiting toxicities occurred in the 15 subjects at dose levels 0, 1 or 2. Pharmacokinetic analyses showed mean peak plasma concentrations of regadenoson for dose levels 0, 1 and 2 were 0.37 ng/ml, 1.16 ng/ml and 2.19 ng/ml, respectively. Based on animal models, we suspect that biologically active plasma concentrations are approximately 1 ng/ml. At baseline, iNKT cells were activated in patients with SCD as defined by elevated levels of phospho-NF-kB expression and A2AR expression as assessed by anti-A2AR immunoreactivity. In particular, CD4+ iNKT cells showed increased phospho-NF-kB expression whereas CD4- iNKT cells were not activated. At dose levels 1 and 2, regadenoson decreased iNKT cell activation in patients with SCD. For example, when pre- and post-12 hour regadenoson infusion measurements were compared at dose level 2 (N=5), mean percent of cells expressing phospho-NF-kB in the activation gate was 40% (90% CI 23–58) at baseline and decreased by 53% at the end of infusion to 19% (90% CI 6–33). Similar results were found in measurements of A2ARs. We conclude that infusional regadenoson administered at 0.6 and 1.44 mcg/kg/hour is safe during a 12 hour infusion and decreases activation of iNKT cells. Our next steps are to determine whether infusions of a longer duration (24 and 48 hours) are safe and similarly biologically effective during a VOC. We then intend to conduct a clinical trial to measure the clinical efficacy of regadenoson infusion during VOC and acute chest syndrome episodes. Disclosures: Off Label Use: Regadenoson is an adenosine 2A receptor agonist that is FDA approved for use during myocardial stress imaging. We are examining the safety of regadenoson in patients with sickle cell disease. Nathan:Astellas: Research Funding.

Description: Background: Von Willebrand disease (VWD) is the most common inherited bleeding disorder, affecting 1% of the population, and characterized by deficient or defective von Willebrand factor (VWF). Among women with VWD, up to 80% have heavy menstrual bleeding (HMB), many of whom have depleted iron stores and iron deficiency anemia with reduced physical functioning, anxiety, depression, and poor quality of life. HMB is a serious problem causing significant health burden for those affected. The lack of effective therapies for menorrhagia is a major unmet healthcare need in women with VWD: in up to 30% desmopressin (DDAVP), combined oral contraceptives (COCs) hormones, or the recommended non-hormonal agent, tranexamic acid (Lysteda®, TA) may be ineffective or poorly tolerated. VWF concentrates, including plasma-derived VWF (pdVWF, Humate-P®) and recombinant VWF (rVWF, Vonvendi®) safely reduce bleeds in VWD, but few data exist on VWF use in menorrhagia, and no prospective trials are available to guide treatment. As rVWF has higher purity, potency, and a longer half-life than pdVWF, this phase III trial will compare rVWF with TA in reducing menorrhagia in women with type 1 VWD. Methods: This is an NHLBI-funded U01 phase III multicenter, prospective, randomized, crossover trial in to compare IV rVWF vs. po TA in reducing menorrhagia in type 1 VWD, clinicaltrials.gov, NCT02606045. Women with type 1 VWD, VWF:RCo100 in at least one of the last two cycles, are eligible. Exclusions include hypothyroidism, past thrombosis, and renal disease. Subjects are randomized to rVWF 40 IU/kg IV day 1 vs. TA 1300 mg po three times daily days 1-5 in each of two consecutive cycles. The order of treatment is determined by randomization: in Group 1, rVWF is given in cycles 1 and 2, and TA in cycles 3 and 4; while in Group 2, TA is given in cycles 1 and 2, and rVWF in cycles 3 and 4. A rescue dose day of rVWF 40 IU/kg may be given day 2 of cycles in which rVWF is given. The primary endpoint is a 40-point reduction in PBAC, a validated measure of menstrual loss, after 2 cycles. As rVWF is a greater burden (IV, cost), to show it is superior to TA, it should improve PBAC 40 points more from baseline than TA. Secondary endpoints are cycle severity, cycle length, QoL (SF-36, Ruta, CDC-HRQ0L-14, CES-D), and satisfaction survey. Treatment response will also be compared with VWF assays and VWF genotype. Safety is assessed by number of rescue doses, other bleeding, thrombosis, and allergic reaction. Our research hypothesis is that rVWF will be superior, producing a greater improvement, by at least 40 points, in PBAC, than TA. We also hypothesize that rVWF will be as safe, tolerable, and acceptable as TA, and that VWF assays and VWF genotype will predict response to treatment. A sample size of 60 (inflated to 66 for 5% attrition) will provide 84% power to detect a difference in improvement of 40 points between rVWF and TA. Analysis will be by intent-to-treat analyses, with a two-tailed alternative hypothesis with type 1 error rate of 0.05, a 4-period 2-group (AABB/BBAA) crossover design, and an estimated between-subject standard deviation (SD) of 63 points and within subject SD of 100 points. Results: A total of 442 potential subjects have been identified at 19 participating HTCs, of whom 33 (7.5%) are eligible, and 2 enrolled. The most common reason for ineligibility is use of an IUD (15.6%), COCs (9.4%), age

Description: Background Multiple lines of evidence suggest that invariant NKT (iNKT) cells generate an inflammatory cascade that promotes and sustains sickle cell vaso-occlusion. In prior studies of mouse models and patients with sickle cell disease (SCD), iNKT cells are increased in number and more likely to be activated compared to controls. Depleting iNKT cells in a mouse model of SCD decreases inflammation and prevents end-organ injury. NKTT120 is a humanized monoclonal antibody that specifically depletes iNKT cells. Preclinical studies show that NKTT120 has high affinity and specificity for iNKT cells. NKTT120 depletes iNKT cells in a dose-dependent manner. Return of iNKT cells to the peripheral circulation following NKTT120 administration occurs in a dose- and time-dependent manner. Our global hypothesis is that NKTT120 will deplete iNKT cells, reduce inflammation and prevent painful vaso-occlusive crises. In this phase 1 dose-escalation study, we will examine the safety of NKTT120 in steady state adults with SCD. Objective To determine the safety, maximum tolerated dose (MTD), pharmacokinetics, and pharmacodynamics of NKTT120 in steady state adults with SCD. The optimal dose for a phase 2 study of NKTT120 will deplete iNKT for approximately 3 months allowing for periodic dosing. Methods Phase 1 study utilizing a 3+3 design to evaluate single doses escalated over a range from 0.001 mg/kg to 0.1 mg/kg (0.001, 0.003, 0.01, 0.03, and 0.10 mg/kg). Primary outcome measure is safety. Secondary outcomes include pain, analgesic use, quality of life (QoL), and pulmonary function. During a screening run-in period and after dosing of NKTT120, subjects will maintain a daily smartphone eDiary (eSCaPe) to report pain, respiratory symptoms and analgesic use. ASCQ-Me and PROMIS QoL questionnaires will be administered at clinic visits. The screening run-in outcomes will be used as baseline comparison for values obtained post-dosing. Results One month of follow-up data on iNKT cell numbers is available for the first four patients in the study (Figure 1). Three subjects received the lowest dose of 0.001 mg/kg and 1 subject received 0.003 mg/kg. All 4 subjects showed a reduction in the iNKT cell percent of CD3+ T cells 6 hours after NKTT120 administration. Three subjects have completed the study with iNKT cells returning to pre-dosing levels at day 7 while the remaining subject is awaiting iNKT cell recovery five weeks after dosing. The effects of NKTT120 were specific to iNKT cells as T cell, B cell and NK cell percent of lymphocytes was not affected. NKTT120 has been well tolerated with no adverse events reported. Conclusions In steady state adults with SCD, NKTT120 administered at the lowest dose of 0.001 mg/kg specifically reduces iNKT cells without toxicity. The dose of 0.003 mg/kg is currently being evaluated in this ongoing trial and higher doses of NKTT120 are anticipated to further deplete iNKT cells in the blood and tissue with longer times to recovery. This reduction of iNKT cells should result in a suppression of the inflammatory stimuli that promote many of the pathophysiologic sequelae seen in SCD. Disclosures: Field: NKT Therapeutics: Consultancy. Eaton:NKT Therapeutics: Employment, Equity Ownership. Mashal:NKT Therapeutics: Employment, Equity Ownership. Nathan:NKT Therapeutics: Consultancy.