ABSTRACT
Invariant natural killer T-lymphocytes (iNKT) are unique immunomodulatory innate T cells with an invariant TCRα recognizing glycolipids presented on MHC class-I-like CD1d molecules. Activated iNKT rapidly secrete pro-and anti-inflammatory cytokines, potentiate immunity, and modulate inflammation. Here, we report the effects of in vivo iNKT activation in Mauritian-origin cynomolgus macaques by a humanized monoclonal antibody, NKTT320, that binds to the invariant region of the iNKT TCR. NKTT320 led to rapid iNKT activation, increased polyfunctionality, and elevation of multiple plasma analytes within 24 hours. Flow cytometry and RNA-Seq confirmed downstream activation of multiple immune subsets, enrichment of JAK/STAT and PI3K/AKT pathway genes, and upregulation of inflammation-modulating genes. NKTT320 also increased iNKT frequency in adipose tissue and did not cause iNKT anergy. Our data indicate that NKTT320 has a sustained effect on in vivo iNKT activation, potentiation of innate and adaptive immunity, and resolution of inflammation, which supports its future use as an immunotherapeutic.
ABSTRACT
Invariant natural killer T (iNKT) cells are innate-like T lymphocytes characterized by the expression of an invariant T cell receptor (iTCR) that recognizes glycolipid antigens presented by the MHC I-like CD1d molecule. Following antigenic stimulation, iNKT cells rapidly produce large amounts of cytokines that can trans-activate dendritic cells (DC) and promote the anti-tumor functions of cytotoxic lymphocytes, such as natural killer (NK) and CD8 T cells. Additionally, iNKT cells can mediate robust and direct cytotoxicity against CD1d+ tumor targets. However, many tumors down-regulate CD1d and evade iNKT cell attack. To circumvent this critical barrier to iNKT cell anti-tumor activity, a novel monoclonal antibody (mAb), NKT14 has been recently developed. This agonistic antibody binds directly and specifically to the iTCR of murine iNKT cells. In the current study, we demonstrate that NKT14m mediates robust activation, cytokine production and degranulation of murine iNKT cells, in vitro. Consistently, NKT14m also promoted iNKT cell activation and immunomodulatory functions, in vivo. Finally, administration of NKT14m with low dose interleukin (IL)-12 further augmented iNKT cell IFN-γ production in vivo, and this combination conferred superior suppression of tumor cell growth compared to NKT14m or IL-12 alone. Together, these data demonstrate that a combination treatment consisting of low dose IL-12 and iTCR-specific mAb may be an attractive alternative to activate iNKT cell anti-tumor functions.
Subject(s)
Antibodies, Monoclonal/pharmacology , Drug Therapy, Combination/methods , Interferon-gamma/metabolism , Interleukin-12/pharmacology , Lymphoma/drug therapy , Natural Killer T-Cells/immunology , Receptors, Antigen, T-Cell/immunology , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/immunology , Antigens, CD1d/immunology , Cell Line, Tumor , Cytokines/metabolism , Cytotoxicity, Immunologic/drug effects , Drug Synergism , Immunomodulation/drug effects , Lymphoma/immunology , Mice , Mice, Inbred C57BL , Natural Killer T-Cells/drug effects , Natural Killer T-Cells/metabolismABSTRACT
Invariant natural killer T cells (iNKTs) directly kill tumor cells and trans-activate the anti-tumor functions of dendritic cells (DC), natural killer (NK) cells, and T and B cells. As such, iNKTs serve as a powerful tool for use in cell-based cancer immunotherapy. iNKT cell activation commonly requires engagement of the invariant T cell receptor (iTCR) by CD1d presenting glycolipid antigens. However, transformed cells often down-regulate CD1d expression, which results in a reduction of iNKT cell anti-tumor functions. One approach to circumvent this critical barrier to iNKT cell activation is to develop an agonistic antibody that binds directly to the iTCR without the requirement for CD1d-mediated antigen presentation. To this end, we have characterized the iNKT cell stimulatory properties of NKTT320, a novel, recombinant, humanized, monoclonal antibody that binds selectively and with high affinity to human iTCRs. Strikingly, immobilized NKTT320 mediated robust iNKT cell activation (upregulation of CD25 and CD69) and proliferation (carboxyfluorescein succinimidyl ester (CFSE) dilution), as well as Th1 and Th2 cytokine production. Additionally, iNKTs stimulated by plate-bound NKTT320 exhibited increased intracellular levels of granzyme B and degranulation (exposure of CD107 on the cell surface). Furthermore, both soluble and immobilized NKTT320 induced iNKT cell-mediated activation of bystander immune cells, suggesting that this novel anti-iTCR antibody facilitates both direct and indirect iNKT cell cytotoxicity. These studies are significant, as they provide a framework by which iNKT cell anti-cancer functions could be enhanced for therapeutic purposes.
Subject(s)
Antibodies, Monoclonal, Humanized/pharmacology , Lymphocyte Activation/drug effects , Lymphocyte Activation/immunology , Natural Killer T-Cells/drug effects , Natural Killer T-Cells/immunology , Biomarkers , Cell Degranulation/drug effects , Cell Degranulation/immunology , Cell Line, Tumor , Cytokines/metabolism , Dose-Response Relationship, Drug , Histocompatibility Antigens Class II/immunology , Humans , Immunophenotyping , Natural Killer T-Cells/metabolismABSTRACT
Invariant natural killer T (iNKT) cells are a small population of T lymphocytes that expresses an invariant T cell receptor with a unique specificity for glycolipid antigens. Their activation using the glycolipid α-galactosylceramide (α-GalCer) triggers innate and adaptive immune responses. The use of α-GalCer in preclinical models as a single antitumor treatment showed moderate effect, but its efficacy in cancer patients was less effective. In addition, this glycolipid induces long-term iNKT-cell anergy precluding the possibility of retreatment. Recently, the first murine iNKT-cell agonistic antibody, NKT14m, has been developed. Here, we analyzed, for the first time, the antitumor efficacy of NKT14m in a B-cell lymphoma model. In a therapeutic setting, a single dose of NKT14m had a moderate antitumor efficacy that was associated with an increase of IFN-γ producing iNKT cells even after a second dose of the NKT14m antibody. Importantly, the combination of a single dose of NKT14m with cyclophosphamide had a potent antitumor efficacy and long-lasting immunity in vivo. Our findings provide the first evidence of the in vivo antitumor efficacy of NKT14m antibody, showing that, either alone or in combination with chemotherapy, induces an effective antitumor response. These results open new opportunities for iNKT-cell mediated immunotherapy to treat B-cell lymphoma.
ABSTRACT
iNKT cells and mast cells have both been implicated in the syndrome of allergic asthma through their activation-induced release of Th2 type cytokines and secretion of histamine and other mediators, respectively, which can promote airways hyperresponsiveness (AHR) to agents such as methacholine. However, a mechanistic link between iNKT cells and mast cell recruitment or activation has never been explored. Our objective was to determine whether iNKT cells are necessary for the recruitment of mast cells and if iNKT cells can influence the acute allergen induced bronchoconstriction (AIB) caused by mast cell mediator release. To do so, we pharmacologically eliminated iNKT cells using a specific antibody (NKT-14) and examined its impact on airway inflammation and physiological phenotype. In mice treated with NKT-14, the elimination of iNKT cells was sufficient to prevent AHR and pulmonary eosinophilic inflammation elicited by administration of the iNKT cell agonist αGalCer. In mice treated with NKT-14 and then sensitized and challenged with house dust mite extract (HDM), eliminating the iNKT cells significantly reduced both AHR and AIB but did not affect pulmonary inflammation, the mast cell population, nor the release of the mast cell mediators mast cell protease-1 and prostaglandin D2. We conclude that while iNKT cells contribute to the phenotype of allergic airways disease through the manifestation of AIB and AHR, their presence is not required for mast cell recruitment and activation, or to generate the characteristic inflammatory response subsequent to allergen challenge.
Subject(s)
Bronchoconstriction/immunology , Mast Cells/metabolism , Natural Killer T-Cells/metabolism , Respiratory Hypersensitivity/immunology , Allergens/immunology , Animals , Chymases/metabolism , Disease Models, Animal , Eosinophils/metabolism , Female , Hypersensitivity/immunology , Inflammation/immunology , Lung/immunology , Lung/pathology , Mast Cells/immunology , Mice , Mice, Inbred BALB C , Natural Killer T-Cells/immunology , Phenotype , Prostaglandin D2/metabolism , Pyroglyphidae/immunologyABSTRACT
Invariant NKT (iNKT) cells can be activated to stimulate a broad inflammatory response. In murine models of sickle cell disease (SCD), interruption of iNKT cell activity prevents tissue injury from vaso-occlusion. NKTT120 is an anti-iNKT cell monoclonal antibody that has the potential to rapidly and specifically deplete iNKT cells and, potentially, prevent vaso-occlusion. We conducted an open-label, multi-center, single-ascending-dose study of NKTT120 to determine its pharmacokinetics, pharmacodynamics and safety in steady-state patients with SCD. Doses were escalated in a 3+3 study design over a range from 0.001 mg/kg to 1.0 mg/kg. Twenty-one adults with SCD were administered NKTT120 as part of 7 dose cohorts. Plasma levels of NKTT120 predictably increased with higher doses. Median half-life of NKTT120 was 263 hours. All subjects in the higher dose cohorts (0.1 mg/kg, 0.3 mg/kg, and 1 mg/kg) demonstrated decreased iNKT cells below the lower limit of quantification within 6 hours after infusion, the earliest time point at which they were measured. In those subjects who received the two highest doses of NKTT120 (0.3, 1 mg/kg), iNKT cells were not detectable in the peripheral blood for a range of 2 to 5 months. There were no serious adverse events in the study deemed to be related to NKTT120. In adults with SCD, NKTT120 produced rapid, specific and sustained iNKT cell depletion without any infusional toxicity or attributed serious adverse events. The next step is a trial to determine NKTT120's ability to decrease rate of vaso-occlusive pain episodes. TRIAL REGISTRATION: clinicaltrials.gov NCT01783691.
Subject(s)
Anemia, Sickle Cell/immunology , Anemia, Sickle Cell/therapy , Antibodies, Monoclonal, Humanized/therapeutic use , Lymphocyte Depletion/methods , Natural Killer T-Cells/immunology , Adolescent , Adult , Animals , Antibodies, Monoclonal, Humanized/administration & dosage , Antibodies, Monoclonal, Humanized/pharmacokinetics , Child , Cohort Studies , Female , Half-Life , Humans , Male , Young AdultABSTRACT
During toxic shock syndrome (TSS), bacterial superantigens trigger a polyclonal T -cell response leading to a potentially catastrophic "cytokine storm". Whether innate-like invariant natural killer T (iNKT) cells, with remarkable immunomodulatory properties, participate in TSS is unclear. Using genetic and cell depletion approaches, we generated iNKT cell-deficient, superantigen-sensitive HLA-DR4-transgenic (DR4tg) mice, which were compared with their iNKT-sufficient counterparts for responsiveness to staphylococcal enterotoxin B (SEB). Both approaches indicate that iNKT cells are pathogenic in TSS. Importantly, treating DR4tg mice with a TH2-polarizing glycolipid agonist of iNKT cells reduced SEB-inflicted morbidity/mortality. Therefore, iNKT cells may constitute an attractive therapeutic target in superantigen-mediated illnesses.
Subject(s)
HLA-DR4 Antigen/genetics , Natural Killer T-Cells/immunology , Shock, Septic/immunology , Shock, Septic/prevention & control , Animals , Disease Models, Animal , Enterotoxins/immunology , Humans , Mice , Mice, Inbred C57BL , Mice, Transgenic , Superantigens/blood , Superantigens/immunologyABSTRACT
Invariant Natural Killer T (iNKT) cells are a T cell subset expressing an invariant T Cell Receptor (TCR) that recognizes glycolipid antigens rather than peptides. The cells have both innate-like rapid cytokine release, and adaptive-like thymic positive selection. iNKT cell activation has been implicated in the pathogenesis of allergic asthma and inflammatory diseases, while reduced iNKT cell activation promotes infectious disease, cancer and certain autoimmune diseases such as Type 1 diabetes (T1D). Therapeutic means to reduce or deplete iNKT cells could treat inflammatory diseases, while approaches to promote their activation may have potential in certain infectious diseases, cancer or autoimmunity. Thus, we developed invariant TCR-specific monoclonal antibodies to better understand the role of iNKT cells in disease. We report here the first monoclonal antibodies specific for the mouse invariant TCR that by modifying the Fc construct can specifically deplete or activate iNKT cells in vivo in otherwise fully immuno-competent animals. We have used both the depleting and activating version of the antibody in the NOD model of T1D. As demonstrated previously using genetically iNKT cell deficient NOD mice, and in studies of glycolipid antigen activated iNKT cells in standard NOD mice, we found that antibody mediated depletion or activation of iNKT cells respectively accelerated and retarded T1D onset. In BALB/c mice, ovalbumin (OVA) mediated airway hyper-reactivity (AHR) was abrogated with iNKT cell depletion prior to OVA sensitization, confirming studies in knockout mice. Depletion of iNKT cells after sensitization had no effect on AHR in the conducting airways but did reduce AHR in the lung periphery. This result raises caution in the interpretation of studies that use animals that are genetically iNKT cell deficient from birth. These activating and depleting antibodies provide a novel tool to assess the therapeutic potential of iNKT cell manipulation.
Subject(s)
Antibodies, Monoclonal, Murine-Derived/pharmacology , Asthma/immunology , Diabetes Mellitus, Type 1/immunology , Natural Killer T-Cells/immunology , Receptors, Antigen, T-Cell/antagonists & inhibitors , Animals , Antibodies, Monoclonal, Murine-Derived/immunology , Asthma/genetics , Asthma/pathology , Asthma/therapy , Diabetes Mellitus, Type 1/genetics , Diabetes Mellitus, Type 1/pathology , Lymphocyte Depletion/methods , Mice , Mice, Inbred BALB C , Mice, Inbred NOD , Natural Killer T-Cells/pathology , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/immunologyABSTRACT
OBJECTIVE: Aptamers are oligonucleotides targeting protein-protein interactions with pharmacokinetic profiles and activity reversal options. Although P-selectin and von Willebrand factor (vWF) have been implicated in the development of venous thrombosis (VT), no studies have directly compared aptamer efficacy with standard of care in VT. In this study, ARC5692, an anti-P-selectin aptamer, and ARC15105, an anti-vWF aptamer, were compared with low-molecular-weight heparin, enoxaparin, to test the efficacy of P-selectin or vWF inhibition in promoting thrombus resolution and preventing vein wall fibrosis, in a baboon model of VT. APPROACH AND RESULTS: Groups were as follows: treatment arm: animals received P-selectin or vWF aptamer inhibitors or enoxaparin (n=3 per group). Controls received no treatment (n=3). Prophylactic arm: animals received P-selectin inhibitor (n=4) or vWF inhibitor (n=3). Treatment arm: P-selectin-inhibitor demonstrated a significant improvement in vein recanalization by magnetic resonance venography (73% at day 21), and significantly decreased vein wall collagen, compared with all groups. Anti-P-selectin equaled enoxaparin in maintaining valve competency by ultrasound. All control animals had compromised valve competency post thrombosis. Prophylactic arm: animals receiving P-selectin and vWF inhibitors demonstrated improved vein recanalization by magnetic resonance venography versus controls (80% and 85%, respectively, at day 21). Anti-P-selectin protected iliac valve function better than anti-vWF, and both improved valve function versus controls. No adverse bleeding events were observed. CONCLUSIONS: The P-selectin inhibitor aptamer promoted iliac vein recanalization, preserved valve competency, and decreased vein wall fibrosis. The results of this work suggest that P-selectin inhibition maybe an ideal target in the treatment and prophylaxis of deep VT, warranting clinical trials.
Subject(s)
Aptamers, Nucleotide/pharmacology , Enoxaparin/pharmacology , Fibrinolytic Agents/pharmacology , Iliac Vein/drug effects , P-Selectin/antagonists & inhibitors , Venous Thrombosis/prevention & control , von Willebrand Factor/antagonists & inhibitors , Animals , Blood Coagulation/drug effects , Collagen/metabolism , Disease Models, Animal , Fibrin/metabolism , Fibrosis , Iliac Vein/diagnostic imaging , Iliac Vein/metabolism , Iliac Vein/pathology , Leukocytes/drug effects , Leukocytes/metabolism , Magnetic Resonance Angiography , P-Selectin/metabolism , Papio , Phlebography/methods , Platelet Aggregation/drug effects , Time Factors , Ultrasonography , Venous Thrombosis/diagnostic imaging , Venous Thrombosis/metabolism , Venous Thrombosis/pathology , Venous Valves/drug effects , Venous Valves/metabolism , Venous Valves/pathology , von Willebrand Factor/metabolismABSTRACT
BACKGROUND: In recent years, a number of tissue factor pathway inhibitor (TFPI) antagonists have been developed to serve as bypassing agents to improve hemostasis in hemophilia A. Since TFPI antagonists and FVIII concentrates are procoagulants, their combined effect on spatial clot formation could be potentially pro-thrombotic. OBJECTIVE: To investigate the cooperative effect of TFPI inhibition and supplementation of FVIII in hemophilia A in a spatial, reaction-diffusion experiment in vitro. METHODS: Plasma was collected at different time points from hemophilia A patients undergoing prophylaxis and was supplemented in vitro with TFPI inhibitor BAX499 (formerly ARC19499) at concentrations from 0 up to 600nM. Clotting propagation in recalcified plasma activated by a surface with immobilized tissue factor (TF) was monitored by videomicroscopy. RESULTS: Increasing concentration of BAX499 improved coagulation for all hemophilia A plasma samples activated with TF at 1.6pmole/m(2) by shortening lag time and increasing initial clot growth velocity and clot size. In contrast, plasma concentration of FVIII had little effect on lag time, but increased spatial clot growth velocity. There was a decrease in the BAX499 efficiency as FVIII concentration increased (lag time shortened by 50% if FVIII:C<5%, but the effect was only 25% if FVIII:C>30%). CONCLUSIONS: The results indicate that BAX499 has an effect on clotting in hemophilia A plasma at low FVIII concentrations, however has little effect at high FVIII concentrations.
Subject(s)
Aptamers, Nucleotide/pharmacology , Factor VIII/pharmacology , Fibrin/metabolism , Hemophilia A/blood , Hemophilia A/drug therapy , Aptamers, Nucleotide/pharmacokinetics , Drug Interactions , Factor VIII/pharmacokinetics , Humans , Lipoproteins/antagonists & inhibitorsABSTRACT
Invariant Natural Killer T (iNKT) cells are a subset of T cells recognizing glycolipid antigens presented by CD1d. Human iNKT cells express a conserved T cell receptor (TCR)-α chain (Vα24-Jα18) paired with a specific beta chain, Vß11. The cells are both innate-like, with rapid cytokine release, and adaptive-like, including thymic positive selection. Over activation of iNKT cells can mediate tissue injury and inflammation in multiple organ systems and play a role in mediating the pathology associated with clinically important inflammatory diseases. At the same time, iNKT cell activation can play a role in protecting against infectious disease and cancer or modulate certain autoimmune diseases through its impact on both the innate and adaptive immune system. This suggests that approaches to cause iNKT cell reduction and/or depletion could treat inflammatory diseases while approaches to promote activation may have therapeutic potential in certain infections, cancer or autoimmune disease. This report summarizes the characterization of a humanized monoclonal depleting antibody (NKTT120) in the cynomolgus macaque. NKTT120 is being developed to treat iNKT mediated inflammation that is associated with chronic inflammatory conditions like sickle cell disease and asthma. NKTT120 binds to human iTCRs and to FCγRI and FCγRIII and has been shown to kill target cells in an ADCC assay at low concentrations consistent with the FCγR binding. iNKT cells were depleted within 24 hours in cynomolgus macaques, but T cell, B cell, and NK cell frequencies were unchanged. iNKT cell recovery was dose and time dependent. T cell dependent antigen responses were not impaired by NKTT120 mediated iNKT depletion as measured by response to KLH challenge. NKTT120 administration did not induce an inflammatory cytokine release at doses up to 10 mg/kg. These data support the use of NKTT120 as an intervention in inflammatory diseases where iNKT reduction or depletion could be beneficial.
Subject(s)
Antibodies, Monoclonal, Humanized/immunology , Antibody Specificity , Natural Killer T-Cells/immunology , Animals , Dose-Response Relationship, Radiation , Female , Humans , Inflammation/immunology , Macaca fascicularisABSTRACT
OBJECTIVE: We investigated the stability, pharmacokinetic, and pharmacodynamic profile of the 2(nd) generation anti-von Willeband factor aptamer ARC15105. METHODS AND RESULTS: Platelet plug formation was measured by collagen/adenosine diphosphate-induced closure time with the platelet function analyzer-100 and platelet aggregation by multiple electrode aggregometry. Platelet adhesion was measured on denuded porcine aortas and in a flow chamber. Aptamer stability was assessed by incubation in nuclease rich human, monkey, and rat serum for up to 72 hours. Pharmacokinetic and pharmacodynamic profiles were tested in cynomolgus monkeys after IV and SC administration. The median IC(100) and IC(50) to prolong collagen/adenosine diphosphate-induced closure timewere 27 nmol/L and 12 nmol/L, respectively. ARC15105 (1.3 µmol/L) completely inhibited ristocetin-induced platelet aggregation in whole blood (P<0.001), but also diminished collagen, ADP, arachidonic acid, and thrombin receptor activating peptide-induced platelet aggregation to some extent (P<0.05). ARC15105 (40 nmol/L) decreased platelet adhesion by >90% on denuded porcine aortas (P<0.001), which was comparable to the degree of inhibition obtained with abciximab. ARC15105 (100 nmol/L) also inhibited platelet adhesion to collagen under arterial shear in a flow chamber by >90% (P<0.001). The IV and SC terminal half-lives in cynomolgus monkeys were 67 and 65 hours, respectively, and the SC bioavailability was ≈98%. Allometric scaling estimates the human T(1/2) would be ≈217 hours. Pharmacodynamic analysis confirms that ARC15105 inhibits von Willeband factor activity >90% in blood samples taken 300 hours after a 20 mg/kg IV or SC dose in monkeys. CONCLUSIONS: The potency, pharmacokinetic profile, and SC bioavailability of ARC15105 support its clinical investigation for chronic inhibition of von Willeband factor -mediated platelet activation.
Subject(s)
Aptamers, Nucleotide/therapeutic use , Aptamers, Peptide/pharmacology , Blood Platelets/drug effects , Myocardial Infarction/blood , Platelet Activation/drug effects , Platelet Adhesiveness/drug effects , Platelet Aggregation Inhibitors/pharmacology , von Willebrand Factor/antagonists & inhibitors , Aged , Animals , Aptamers, Nucleotide/administration & dosage , Aptamers, Nucleotide/pharmacokinetics , Aptamers, Peptide/administration & dosage , Aptamers, Peptide/pharmacokinetics , Austria , Biological Availability , Blood Platelets/metabolism , Boston , Case-Control Studies , Collagen/metabolism , Cross-Sectional Studies , Dose-Response Relationship, Drug , Drug Stability , Female , Half-Life , Humans , Injections, Intravenous , Injections, Subcutaneous , Macaca fascicularis , Male , Middle Aged , Platelet Aggregation Inhibitors/administration & dosage , Platelet Aggregation Inhibitors/pharmacokinetics , Platelet Function Tests , Protein Binding , Quebec , Rats , Swine , von Willebrand Factor/metabolismABSTRACT
Hemophilia is a family of rare bleeding disorders. The two primary types, hemophilia A and hemophilia B, are caused by recessive X-chromosome linked mutations that result in deficiency of coagulation factor VIII (FVIII) or factor IX (FIX), respectively. Clinically, hemophilia is manifested by spontaneous bleeding, particularly into the joints (haemarthrosis) and soft tissue, and excessive bleeding following trauma or surgery. The total overall number of hemophilia patients worldwide is approximately 400,000, however only about 100,000 of these individuals are treated. The first treatment of hemophilia was initiated when it was determined that the clotting deficiency could be corrected by a plasma fraction taken from normal blood. The discovery of factor VIII enrichment by cryoprecipitation of plasma opened a new era of therapy which eventually led to the production of factor concentrates and the subsequent development of highly purified forms of plasma factors. The most significant improvements have been the availability of recombinant forms of factors VIII and IX. Unfortunately, recombinant factors still retain some of the limitations of plasma concentrates. These limitations include development of antibody responses in patients and the relatively short half-life of the molecules requiring frequent injection to maintain effective concentration. Treatment beyond replacement of native factors has been tried. They include the development of modified factor VIII and IX molecules with improved potency, stability and circulating half-life and enhancement of a prothrombotic responses and/or stabilization of coagulation factors via inhibition of key negative regulatory pathways. These approaches will be reviewed in this commentary.
Subject(s)
Blood Coagulation Factors/urine , Hemophilia A/drug therapy , Animals , Factor IX/therapeutic use , Factor VIII/therapeutic use , Factor VIIa/therapeutic use , Factor Xa/genetics , Humans , Lipoproteins/antagonists & inhibitors , Protein C Inhibitor/therapeutic use , Recombinant Proteins/therapeutic useABSTRACT
Hemophilia A and B are caused by deficiencies in coagulation factor VIII (FVIII) and factor IX, respectively, resulting in deficient blood coagulation via the intrinsic pathway. The extrinsic coagulation pathway, mediated by factor VIIa and tissue factor (TF), remains intact but is negatively regulated by tissue factor pathway inhibitor (TFPI), which inhibits both factor VIIa and its product, factor Xa. This inhibition limits clot initiation via the extrinsic pathway, whereas factor deficiency in hemophilia limits clot propagation via the intrinsic pathway. ARC19499 is an aptamer that inhibits TFPI, thereby enabling clot initiation and propagation via the extrinsic pathway. The core aptamer binds tightly and specifically to TFPI. ARC19499 blocks TFPI inhibition of both factor Xa and the TF/factor VIIa complex. ARC19499 corrects thrombin generation in hemophilia A and B plasma and restores clotting in FVIII-neutralized whole blood. In the present study, using a monkey model of hemophilia, FVIII neutralization resulted in prolonged clotting times as measured by thromboelastography and prolonged saphenous-vein bleeding times, which are consistent with FVIII deficiency. ARC19499 restored thromboelastography clotting times to baseline levels and corrected bleeding times. These results demonstrate that ARC19499 inhibition of TFPI may be an effective alternative to current treatments of bleeding associated with hemophilia.
Subject(s)
Aptamers, Nucleotide/pharmacology , Blood Coagulation/drug effects , Hemostasis/drug effects , Lipoproteins/antagonists & inhibitors , Animals , Aptamers, Nucleotide/chemistry , Bleeding Time , Disease Models, Animal , Factor VIII/metabolism , Factor VIIa/metabolism , Factor Xa/metabolism , Hemophilia A/blood , Hemophilia A/drug therapy , Hemophilia B/blood , Hemophilia B/drug therapy , Humans , In Vitro Techniques , Macaca fascicularis , Recombinant Proteins/antagonists & inhibitors , Thrombin/biosynthesis , Thromboplastin/metabolismABSTRACT
The von Willebrand factor (vWF) aptamer, ARC1779 that blocks the binding of vWF A1-domain to platelet glycoprotein 1b (GPIb) at high shear, may deliver a site-specific antithrombotic effect. We investigated the efficiency of ARC1779 on platelet function in patients with coronary artery disease (CAD) on double antiplatelet therapy. Blood from patients taking aspirin and clopidogrel and from normal volunteers was treated ex vivo with ARC1779 or abciximab, either prior to perfusion (pretherapy) or 10 minutes following the initiation of perfusion (posttherapy) on damaged arteries. Under pre- but not posttherapy, platelet adhesion was significantly reduced by ARC1779 at 83 and 250 nmol/L and by abciximab (100 nmol/L) versus placebo (4.8, 3.8, and 2.9 vs 7.3 platelets × 10(6)/cm(2), P < .05). In contrast to abciximab, ARC1779 did not significantly affect platelet aggregation, P-selectin expression, and platelet-leukocyte binding. These proof-of-concept data may constitute the framework for randomized clinical investigations of this novel antiplatelet therapy among patients with CAD.
Subject(s)
Aptamers, Nucleotide/therapeutic use , Aspirin/therapeutic use , Coronary Artery Disease/drug therapy , Platelet Adhesiveness/drug effects , Platelet Aggregation Inhibitors/therapeutic use , Thrombosis/drug therapy , Ticlopidine/analogs & derivatives , Abciximab , Adult , Antibodies, Monoclonal/therapeutic use , Clopidogrel , Coronary Artery Disease/blood , Female , Humans , Immunoglobulin Fab Fragments/therapeutic use , Male , Middle Aged , Platelet Activation/drug effects , Platelet Aggregation/drug effects , Thrombosis/blood , Ticlopidine/therapeutic use , von Willebrand Factor/antagonists & inhibitors , von Willebrand Factor/metabolismABSTRACT
Adhesive interactions between circulating sickle red blood cells (RBCs), leukocytes, and endothelial cells are major pathophysiologic events in sickle cell disease (SCD). To develop new therapeutics that efficiently inhibit adhesive interactions, we generated an anti-P-selectin aptamer and examined its effects on cell adhesion using knockout-transgenic SCD model mice. Aptamers, single-stranded oligonucleotides that bind molecular targets with high affinity and specificity, are emerging as new therapeutics for cardiovascular and hematologic disorders. In vitro studies found that the anti-P-selectin aptamer exhibits high specificity to mouse P-selectin but not other selectins. SCD mice were injected with the anti-P-selectin aptamer, and cell adhesion was observed under hypoxia. The anti-P-selectin aptamer inhibited the adhesion of sickle RBCs and leukocytes to endothelial cells by 90% and 80%, respectively. The anti-P-selectin aptamer also increased microvascular flow velocities and reduced the leukocyte rolling flux. SCD mice treated with the anti-P-selectin aptamer demonstrated a reduced mortality rate associated with the experimental procedures compared with control mice. These results demonstrate that anti-P-selectin aptamer efficiently inhibits the adhesion of both sickle RBCs and leukocytes to endothelial cells in SCD model mice, suggesting a critical role for P-selectin in cell adhesion. Anti-P-selectin aptamer may be useful as a novel therapeutic agent for SCD.
Subject(s)
Anemia, Sickle Cell/physiopathology , Aptamers, Nucleotide/pharmacology , Cell Adhesion/drug effects , Leukocyte Rolling/drug effects , P-Selectin/antagonists & inhibitors , Animals , Disease Models, Animal , Mice , Mice, Inbred C57BL , Mice, Knockout , SELEX Aptamer Technique , Surface Plasmon ResonanceABSTRACT
Previously, we reported the discovery of PSI-697 (1a), a C-2 benzyl substituted quinoline salicylic acid-based P-selectin inhibitor. It is active in a variety of animal models of cardiovascular disease. Compound 1a has also been shown to be well tolerated and safe in healthy volunteers at doses of up to 1200 mg in a phase 1 single ascending dose study. However, its oral bioavailability was low. Our goal was to identify a back up compound with equal potency, increased solubility, and increased exposure. We expanded our structure-activity studies in this series by branching at the alpha position of the C-2 benzyl side chain and through modification of substituents on the carboxylic A-ring of the quinoline. This resulted in discovery of PSI-421 with marked improvement in aqueous solubility and pharmacokinetic properties. This compound has shown oral efficacy in animal models of arterial and venous injury and was selected as a preclinical development compound for potential treatment of such diseases as atherosclerosis and deep vein thrombosis.
Subject(s)
Carotid Artery Injuries/drug therapy , Hydroxyquinolines/chemical synthesis , P-Selectin/antagonists & inhibitors , Salicylates/chemical synthesis , Venous Thrombosis/drug therapy , Administration, Oral , Animals , Caco-2 Cells , Cell Membrane Permeability , Dogs , Drug Stability , Humans , Hydroxyquinolines/pharmacokinetics , Hydroxyquinolines/pharmacology , Leukocyte Rolling/drug effects , Macaca fascicularis , Mice , Mice, Inbred C57BL , Microsomes, Liver/metabolism , Models, Molecular , Papio , Rats , Rats, Sprague-Dawley , Salicylates/chemistry , Salicylates/pharmacology , Solubility , Structure-Activity RelationshipABSTRACT
BACKGROUND: In thrombotic thrombocytopenic purpura (TTP), ultralarge von Willebrand factor (VWF) multimers bind platelet (PLT) glycoprotein Ib and lead to the formation of disseminated fibrin-poor, VWF-rich PLT thrombi. The aptamer ARC1779 blocks binding of the VWF A1 domain to PLT glycoprotein Ib. We evaluated whether ARC1779 inhibits the excessive VWF activity and VWF-mediated PLT function in patients with TTP. STUDY DESIGN AND METHODS: We studied the ex vivo concentration response curves for ARC1779 on PLT function analyzer (PFA-100, Dade Behring) and cone-and-plate analyzer (CPA, Impact-R) PLT function tests, agonist-induced PLT aggregation, and VWF activity of TTP patients (n = 11, three in acute phase and eight in remission) and healthy controls (n = 44). RESULTS: VWF activity and VWF-dependent PLT plug formation were increased in TTP patients relative to healthy controls, but agonist-induced PLT aggregation was not. ARC1779 blocked collagen/adenosine 5'-diphosphate (ADP)-induced PLT plug formation as measured by PFA-100 with an inhibitory concentration (IC)(100) of approximately 1 microg/mL in citrate-anticoagulated samples and approximately 3 to 4 microg/mL in hirudin-anticoagulated samples. A similar concentration of ARC1779 was necessary to block shear-dependent PLT adhesion in both TTP patients and healthy controls using the CPA assay (IC(100) of approx. 1 microg/mL for both). ARC1779 blocked VWF activity with an IC(90) of approximately 3 to 4 microg/mL in all subjects, but did not inhibit PLT aggregation by ADP, collagen, or arachidonic acid even at concentrations much greater than those that fully inhibited VWF-dependent PLT function. CONCLUSIONS: ARC1779 potently and specifically inhibits VWF activity and VWF-dependent PLT function. ARC1779 may be a promising novel therapeutic for the treatment of TTP.
Subject(s)
Blood Platelets/drug effects , Purpura, Thrombotic Thrombocytopenic/drug therapy , von Willebrand Factor/antagonists & inhibitors , Adult , Aptamers, Nucleotide , Blood Platelets/physiology , Cross-Sectional Studies , Female , Humans , Male , Middle Aged , Platelet Aggregation/drug effects , Purpura, Thrombotic Thrombocytopenic/bloodABSTRACT
BACKGROUND: P-selectin antagonism has been shown to decrease thrombogenesis and inflammation in animal models of deep venous thrombosis (DVT). OBJECTIVE: To determine the effectiveness of P-selectin inhibitors versus saline and enoxaparin in venous thrombus resolution in nonhuman primate models of venous thrombosis. METHODS: Studies reporting vein re-opening, inflammation expressed as Gadolinium enhancement and coagulation parameters were searched in the literature and pooled into a meta-analysis using an inverse variance with random effects. RESULTS: Five studies were identified comparing P-selectin/ PSGL-1 inhibitors versus saline or enoxaparin regarding venous thrombosis resolution. Vein re-opening was significantly higher on P-selectin/ PSGL-1 compounds, when compared to saline (Inverse Variance [IV] 95% CI; 44.37 [17.77-70.96], p=0.001, I(2)=97%) and similar to enoxaparin (IV 95% CI; 5.03 [-8.88-18.95], p=0.48, I(2)=41%). Inflammation, reflected as Gadolinium enhancement at magnetic resonance venography (MRV), was significantly decreased in the P-selectin treated group when compared to saline (IV 95% CI; -17.84 [-14.98-(-8.30)], p<0.00001, I(2)=80%). No significant differences on vein wall inflammation were observed between P-selectin/ PSGL-1 inhibitors and enoxaparin treated animals (IV95% CI; -3.59 [-10.67-3.48], p=0.32, I(2)=66%). In addition, there was no differences in the coagulation parameters (aPTT, TCT, BT, D-Dimer, fibrinogen, platelets) between P-selectin/ PSGL-1 inhibitors and enoxaparin (IV 95% CI; -1.12[-2.36-0.11], p=0.07, I(2)=92%), although there was a trend showing less of a prolongation in TCT with P-selectin/PSGL-1 inhibitors compared to enoxaparin (p<0.0001). CONCLUSION: P-selectin antagonism successfully paralleled the low-molecular-weight-heparin enoxaparin, for the treatment of DVT in nonhuman primate models, by decreasing both thrombus burden and inflammation without causing any bleeding complications and without increasing coagulation times.