Two Factor XI Concentrates Correct Impaired Thrombin Generation in Major FXI Deficiency but Are Not Equivalent in Their Effect

Abstract Introduction: Factor XI (FXI) deficiency is an autosomally inherited bleeding disorder characterised by an increased risk of excessive bleeding following trauma or surgery. However, considerable phenotypic heterogeneity of the bleeding tendency is observed between individuals with this disorder. Treatment options for patients requiring FXI replacement are fresh frozen plasma (preferably pathogen-inactivated) or FXI concentrate. Two FXI concentrates are available: Hemoleven® ( LFB Biomedicaments, Les Ulis, France) and Factor XI Concentrate (Bio-Products Laboratory (BPL), Elstree, UK). Guidelines previously recommended a maximum replacement dose of 30 U/Kg for both concentrates but more recently a lower dose of 10-15 U/kg has been advised. The two FXI concentrates may reduce bleeding risk in FIX deficiency following surgery but indications for their use are unclear and treatment in some cases has been associated with thrombosis. Aims: To quantify thrombin generation in major FXI deficiency (FXI:C <15 IU/dL) and to compare the in vitro effects of both FXI concentrates on thrombin generation parameters in this population with each other and with reference range values. To assess the clinical relevance of in vitro results through comparison of thrombin generation following in vitro and in vivo FXI replacement in individuals requiring surgery. Methods: Thrombin generation (TG) was measured in controls (n=50), in individuals with FXI deficiency ( FXI < 15 IU/dL) pre and post in vitro spiking with both FXI concentrates (n=10), and in ex vivo samples following treatment with BPL FXI concentrate (n=3). Blood was drawn into S-Monovette® tubes (Sarstedt, Leicester, U.K.) containing 0.106 M trisodium citrate (1:9, V:V) and corn trypsin inhibitor (CTI) (Haematologic Technologies Inc., Essex Junction, VT, U.S.A.), at a concentration of 20 µg/mL whole blood. Thrombin generation was measured in platelet rich plasma using the Calibrated Automated Thrombography method with a tissue factor trigger of 0.5pM. Statistical analysis was performed using GraphPad prism, version 6 software package (GraphPad, san Diego, CA, USA) using student’s t-test, Mann-Whitney U test or a Wilcoxon signed rank test according to data distribution. P value <0.05 was considered significant. Results: Major FXI deficiency (FXI:C <15 IU/dL) was associated with significantly impaired TG compared to controls, demonstrating reduced endogenous thrombin potential (ETP), peak height and velocity (all p<0.0001) and prolonged time to peak (p = 0.021). All TG parameters significantly improved from baseline with FXI replacement with both concentrates in vitro (equivalent in vivo dose 10 U/Kg). Comparison of the two FXI concentrates demonstrated that LFB Hemoleven® had greater effect on TG than BPL FXI in vitro at all doses (equivalent in vivo doses 10, 20 and 30 U/Kg): higher ETP (p < 0.0001), peak height (p < 0.01) velocity (p < 0.0002) and shorter lag time and time to peak (both p < 0.003). However, some measurements with LFB Hemoleven® exceeded the reference range. At lower doses both FXI concentrates normalised TG parameters in vitro (equivalent in vivo dose 2.5 IU/Kg LFB Hemoleven® or 5 U/Kg BPL FXI). Three patients received in vivo treatment with BPL FXI concentrate prior to surgery. TG was compared between baseline, in vitro spiked and post infusion ex vivo samples. Comparable ETP and peak height results were obtained from in vitro spiked and post infusion ex vivo samples. Conclusions: Individuals with FXI:C levels <15 IU/dL show impaired thrombin generation. Both FXI concentrates improve thrombin generation in vitro in patients with FXI deficiency. However, when tested in vitro with the TG assay, the concentrates differ in potency and dose response and for both concentrates, doses lower than present recommendations normalised thrombin generation. Comparison of in vitro spiked and ex vivo samples suggest that in vitro results could be used to estimate an expected in vivo response to FXI replacement for the BPL product. Acknowledgments: This work is supported by a Fellowship Project Award from the Bayer Hemophilia Awards Program, an unrestricted grant from LFB Biotechnologies and a Wycherley Fellowship grant. Hemoleven® concentrate was kindly provided by LFB Biotechnologies. Disclosures Pike: Bayer: Honoraria, Research Funding; LFB Biotechnologies: Honoraria, Research Funding. Bolton-Maggs:Bio-Products Laboratory (U.K.): Consultancy; LFB Biotechnologies: Consultancy.

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Comparative Studies On Branded Enoxaparin and a US Generic Version of Enoxaparin.

Blood ◽

10.1182/blood.v120.21.2264.2264 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2264-2264

Author(s):

Jeanine M. Walenga ◽

Walter Jeske ◽

Debra Hoppensteadt ◽

Josephine Cunanan ◽

Vicki Escalante ◽

...

Keyword(s):

Molecular Weight ◽

Whole Blood ◽

Research Funding ◽

Thrombin Generation ◽

Ex Vivo ◽

Anticoagulant Effect ◽

Individual Subject ◽

The Us

Abstract Abstract 2264 Background: Low molecular weight heparins (LMWHs) are complex biologic drugs whose heterogeneity in saccharide chain length and in the composition (sulfate, acetyl), content, and location of functional groups can impact their multiple biologic activities. Enoxaparin (Lovenox®) is validated for multiple indications and is the most widely used LMWH in the US. Several generic versions of enoxaparin are currently available in the US. We undertook this study to compare the activity profile of branded and a generic enoxaparin. Methods: Five batches each of branded (B; Sanofi-aventis; Bridgewater, NJ) and generic (G; Sandoz US; Princeton, NJ) enoxaparin were studied. Drugs were purchased through hospital pharmacies as pre-filled syringes containing 40 mg drug. The molecular weight profile of each batch of LMWH was determined using HPLC. To analyze in vitro activities, LMWHs were supplemented to normal human plasma and assessed using amidolytic anti-FXa and anti-FIIa, fibrinokinetic, and thrombin generation assays. Human whole blood was supplemented with LMWHs and the dynamic blood coagulation process was analyzed by thrombelastography (TEG). Whole blood flow cytometry was used to assess the ability of the LMWHs to inhibit tissue factor (TF)-induced platelet activation and lipopolysaccharide (LPS)-induced neutrophil activation. To assess the in vivo effect of the LMWHs, primates treated subcutaneously with a dose of 1 mg/kg LMWH had blood samples drawn pre-treatment and at 4, 6, 12 and 24 hours. Ex vivo pharmacodynamic activities of TFPI release, TAFI inhibition, and thrombin generation inhibition were evaluated. Results: Molecular weight parameters and IC50 values for FXa and FIIa inhibition by branded and generic enoxaparin were comparable. In the in vitro thrombin generation and fibrinokinetic assays, branded enoxaparin exhibited a more potent anticoagulant effect demonstrating slower clot formation with a weaker final clot structure (p=0.01) than generic enoxaparin. Although both the branded and generic enoxaparin produced a concentration-dependent anticoagulant effect in the TEG, there was greater degree of variability for the generic product between blood donors and between batches resulting in a less predictable linear response as drug concentration increased. When the increase in TEG R-time was plotted vs. concentration, branded enoxaparin showed a stronger anticoagulant effect (p=0.05). A concentration-dependent reduction in TF-induced platelet P-selectin expression was observed with branded and generic enoxaparin producing a similar effect. Incubation of whole blood with LPS resulted in a dramatic increase in neutrophil CD11b expression (MFI: 13.1±2.8 vs. 249.0±42.1) which was reduced by increasing concentrations of LMWH. This effect appeared to be stronger for generic than branded enoxaparin (MFI: 165.2±31.9 vs. 208.9±25.9). In primates treated with branded and generic enoxaparin, anti-FXa activity assessed by AUC 0–24hrs was similar. Anti-FIIa activity, however, was significantly higher in primates treated with generic enoxaparin (135±28 vs. 91±20 (μg*hr)/ml; p=0.023). AUC for thrombin generation inhibition was (B) 932±59 vs. (G) 775±119 %inhibition*hr; p=0.029. AUC for TFPI release was (B) 1101±98 vs. (G) 822±13 (ng/*hr)/ml; p=0.006. AUC for inhibition of TAFI activation was (B) 780±73 vs. 906±69 % inhibition*hr; p=0.023. Conclusions: This investigation demonstrated a wider variation in anticoagulant response to generic enoxaparin in comparison to branded enoxaparin. This variation was due to the response of the individual subject as well as to the batch of the product. In addition, both in vitro and in vivo/ex vivo activity differences were observed between branded and generic enoxaparin in several parameters relevant to the antithrombotic effect of LMWH. These findings suggest that simple analytical characterization can establish good quality control in manufacturing but may not assure similarity in biological performance between branded and generic enoxaparin. Thus beside the routinely required characterization, inclusion of additional tests for biologic activities and pharmacodynamic profiling of generic products in animal models may provide useful information on the bioequivalence of the generic versions of enoxaparin. Disclosures: Walenga: Sanofi-Aventis, Paris, France: Research Funding. Jeske:Sanofi-Aventis, Paris, France: Research Funding. Hoppensteadt:Sanofi-Aventis, Paris, France: Research Funding. Cunanan:Sanofi-Aventis, Paris, France: Research Funding. Escalante:Sanofi-Aventis, Paris, France: Research Funding. Khan:Sanofi-Aventis, Paris, France: Research Funding. Bailey:Sanofi-Aventis, Paris, France: Research Funding. Fareed:Sanofi-Aventis, Paris, France: Research Funding. Bakhos:Sanofi-Aventis, Paris, France: Research Funding.

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Cytokine-Mediated Signaling Is Suppressed in Myeloid Cells and Enhanced in Lymphatic Cells in Patients with Chronic Myeloid Leukemia (CML) — Partial Normalization with Tyrosine Kinase Inhibitors.

Blood ◽

10.1182/blood.v114.22.2180.2180 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2180-2180

Author(s):

Sari Jalkanen ◽

Satu Mustjoki ◽

Kimmo Porkka ◽

Jukka Vakkila

Keyword(s):

T Cells ◽

Myeloid Leukemia ◽

Research Funding ◽

Ex Vivo ◽

Healthy Controls ◽

Gm Csf ◽

Single Cell Profiling ◽

Treg Lymphocytes

Abstract Abstract 2180 Poster Board II-157 Introduction. Aberrant phosphorylation of the BCR-ABL1 tyrosine kinase (TK) is characteristic of chronic myeloid leukemia (CML). This oncoprotein interacts directly with intracellular signaling proteins, alters the responsiveness of cytokine receptors and regulates secretion of autocrine cytokines. Targeted inhibition of BCR-ABL1 with TK inhibitor (TKI) imatinib mesylate (IM) is the current standard treatment of CML. For overcoming IM resistance or intolerance, 2nd generation TKIs (nilotinib, dasatinib) with broader kinase inhibition profile have been approved for clinical use. Although in vitro results suggest that TKIs are immunosuppressive, no increases in opportunistic infections or secondary malignancies have been observed to date. In contrast, in some TKI-treated patients immunoactivation in the form of chronic lymphocytosis linked to excellent therapy responses has recently been shown. Dynamic monitoring of aberrant cytokine signaling pathways would aid in understanding and predicting the development of TKI-resistance or adverse/off-target effects. The aim of this study was to analyze the responsiveness of leukocytes to cytokine stimuli in CML patients at diagnosis and during TKI therapy using single-cell profiling of phosphoprotein networks by multiparameter flow cytometry. Patients and methods. The study consisted of 4 healthy controls, 6 CML patients at diagnosis, 6 IM patients and 5 dasatinib patients. Stimuli included GM-CSF, IL-2+IL-10+IFNα and IL-4+IL-6+IFNγ and they were added immeadately to freshly drawn whole blood ex vivo. The readout phosphoproteins were pERK1/2, pSTAT1, pSTAT3, pSTAT5a and pSTAT6 (with isotype controls), and were analyzed separately from granulocytes, monocytes, CD4+ CD25neg T helper cells (Th), CD4neg lymphocytes and CD4+CD25+ T cells including regulatory T-cells (Treg). Analysis was performed with heatmap function of Cytobank software (http://cytobank.stanford.edu/public/). Results. Unstimulated phosphoprotein levels reflecting the activation state of leukocytes in vivo did not differ between healthy controls and CML patients at diagnosis or during dasatinib therapy. Strikingly, in IM patients, baseline levels of pSTAT3 were relatively high indicating in vivo occurring activation of leukocytes in this patient group. We next studied ex vivo responsiveness of immune effector cells with cytokines and found clear differences between healthy controls and CML patients. At CML diagnosis. GM-CSF/pERK1+pSTAT5a, IFNa/pSTAT1,and IL-4/pSTAT6 (stimulus/readout) as well as pSTAT3 responses with all stimuli were suppressed in monocytes. In granulocytes, GM-CSF/pSTAT1 levels were diminished. In Th and Treg lymphocytes, IL-6/pSTAT3 responses were markedly pronounced, while IL-10/pSTAT3 responses were not affected when compared to healthy controls. Such difference was not observed in CD4neg lymphocytes. During TKI therapy. Most patients (9/11) were in cytogenetic remission at the time of analysis. The unresponsiveness of myeloid cells at diagnosis was restored by IM or dasatinib therapy in most, but not all patients. Similarly, in Th and Treg lymphocytes TKI-therapy normalized the enhanced IL-6/pSTAT3 responses that were evident at diagnosis. However, in Th and Treg cells pSTAT3 responses provoked by IL-10 were particularly prominent. Interestingly, one dasatinib patient with aberrant constant blood NK-lymphocytosis and monocytosis had uniquely strong IFNg/pSTAT1 and IL-4/pSTAT6 responses in monocytes. Furthermore, one patient who have stayed in persistent remission after IM discontinuation had exceptionally high pSTAT3 responses with all of stimuli used. Similar kind of signaling profile was unseen with the other patients and could reflect immunoactivation related to leukemia control. Conclusions. Dynamic single-cell profiling of signaling networks is feasible in CML patients and can be used to study mechanisms of aberrant immune reactivity in TKI-treated patients. The method could be particularly suitable for assessing candidate patients for TKI discontinuation. Although in vitro results suggest immunosuppressive effects of TKIs on lymphocytes, leukocytes ex vivo from patients were able to respond similarly to cytokine stimuli as in healthy controls. Disclosures: Mustjoki: BMS: Honoraria. Porkka:BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

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Stroma-Free Ex Vivo Expanded, CD34+ Cord Blood-Derived NK Cells Retain Lytic Activity After Long-Term Engraftment in NSGS Mice

Blood ◽

10.1182/blood.v120.21.580.580 ◽

2012 ◽

Vol 120 (21) ◽

pp. 580-580

Author(s):

Mark Wunderlich ◽

Mahesh Shrestha ◽

Lin Kang ◽

Eric Law ◽

Vladimir Jankovic ◽

...

Keyword(s):

Nk Cells ◽

Research Funding ◽

Nk Cell ◽

Ex Vivo ◽

Employment Equity ◽

Cell Product ◽

Equity Ownership ◽

Cellular Therapeutics

Abstract Abstract 580 Generating a large number of pure, functional immune cells that can be used in human patients has been a major challenge for NK cell-based immunotherapy. We have successfully established a cultivation method to generate human NK cells from CD34+ cells isolated from donor-matched cord blood and human placental derived stem cells, which were obtained from full-term human placenta. This cultivation method is feeder-free, based on progenitor expansion followed by NK differentiation supported by cytokines including thrombopoietin, stem cell factor, Flt3 ligand, IL-7, IL-15 and IL-2. A graded progression from CD34+ hematopoietic progenitor cells (HSC) to committed NK progenitor cells ultimately results in ∼90% CD3-CD56+ phenotype and is associated with an average 10,000-fold expansion achieved over 35 days. The resulting cells are CD16- and express low level of KIRs, indicating an immature NK cell phenotype, but show active in vitro cytotoxicity against a broad range of tumor cell line targets. The in vivo persistence, maturation and functional activity of HSC-derived NK cells was assessed in NSG mice engineered to express the human cytokines SCF, GM-CSF and IL-3 (NSGS mice). Human IL-2 or IL-15 was injected intraperitoneally three times per week to test the effect of cytokine supplementation on the in vivo transferred NK cells. The presence and detailed immunophenotype of NK cells was assessed in peripheral blood (PB), bone marrow (BM), spleen and liver samples at 7-day intervals up to 28 days post-transfer. Without cytokine supplementation, very few NK cells were detectable at any time-point. Administration of IL-2 resulted in a detectable but modest enhancement of human NK cell persistence. The effect of IL-15 supplementation was significantly greater, leading to the robust persistence of transferred NK cells in circulation, and likely specific homing and expansion in the liver of recipient mice. The discrete response to IL-15 versus IL-2, as well as the preferential accumulation in the liver have not been previously described following adoptive transfer of mature NK cells, and may be unique for the HSC-derived immature NK cell product. Following the in vivo transfer, a significant fraction of human CD56+ cells expressed CD16 and KIRs indicating full physiologic NK differentiation, which appears to be a unique potential of HSC-derived cells. Consistent with this, human CD56+ cells isolated ex vivo efficiently killed K562 targets in in vitro cytotoxicity assays. In contrast to PB, spleen and liver, BM contained a substantial portion of human cells that were CD56/CD16 double negative (DN) but positive for CD244 and CD117, indicating a residual progenitor function in the CD56- fraction of the CD34+ derived cell product. The BM engrafting population was higher in NK cultures at earlier stages of expansion, but was preserved in the day 35- cultured product. The frequency of these cells in the BM increased over time, and showed continued cycling based on in vivo BrdU labeling 28 days post-transfer, suggesting a significant progenitor potential in vivo. Interestingly, DN cells isolated from BM could be efficiently differentiated ex vivo to mature CD56+CD16+ NK cells with in vitro cytotoxic activity against K562. We speculate that under the optimal in vivo conditions these BM engrafting cells may provide a progenitor population to produce a mature NK cell pool in humans, and therefore could contribute to the therapeutic potential of the HSC-derived NK cell product. The in vivo activity of HSC-derived NK cells was further explored using a genetically engineered human AML xenograft model of minimal residual disease (MRD) and initial data indicates significant suppression of AML relapse in animals receiving NK cells following chemotherapy. Collectively, our data demonstrate the utility of humanized mice and in vivo xenograft models in characterizing the biodistribution, persistence, differentiation and functional assessment of human HSC-derived cell therapy products, and characterize the potential of HSC-derived NK cells to be developed as an effective off-the-shelf product for use in adoptive cell therapy approaches in AML. Disclosures: Wunderlich: Celgene Cellular Therapeutics: Research Funding. Shrestha:C: Research Funding. Kang:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Law:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Jankovic:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Zhang:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Herzberg:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Abbot:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Hariri:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Mulloy:Celgene Cellular Therapeutics: Research Funding.

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Development of a Human Therapeutic L-Cyst(e)Ine-Degrading Enzyme for the Treatment of Hematological Malignancies

Blood ◽

10.1182/blood.v128.22.1587.1587 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1587-1587

Author(s):

Giulia Agnello ◽

Susan Alters ◽

Joseph Tyler ◽

Jinyun Liu ◽

Peng Huang ◽

...

Keyword(s):

Research Funding ◽

Hematological Malignancies ◽

Ex Vivo ◽

Redox Balance ◽

Lymphocytic Leukemia ◽

Employment Equity ◽

Equity Ownership ◽

Antioxidant Mechanisms

Abstract Cancer cells experience higher intrinsic oxidative stress than their normal counterparts and acquire adaptive antioxidant mechanisms to maintain redox balance. This increased antioxidant capacity has been correlated to malignant transformation, metastasis and resistance to standard anticancer drugs. This enhanced antioxidant state also correlates with cancer cells being more vulnerable to additional oxidative insults, therefore disruption of adaptive antioxidant mechanisms may have significant therapeutic implications. Hematological malignancies including Chronic Lymphocytic Leukemia (CLL), Acute Lymphocytic Leukemia (ALL), Acute Myeloid Leukemia (AML) and Multiple Myeloma (MM) are critically dependent on the cellular antioxidant glutathione (GSH), consistent with the higher intrinsic oxidative stress. L-cysteine is the rate-limiting substrate for GSH biosynthesis and adequate levels of cysteine are critical to maintain the intracellular homeostasis of GSH. CLL and a subset of ALL cells have been reported to rely on the stromal supply of cysteine to increase the synthesis of GSH in order to maintain redox balance, which in turn promotes cell survival and fosters drug resistance. One approach to target this cancer specific dependency is by therapeutic depletion of amino acids via enzyme administration; a clinically validated strategy for the treatment of ALL. Aeglea BioTherapeutics Inc. has developed a bioengineered cysteine and cystine degrading enzyme (Cyst(e)inase, AEB3103) and evaluated its therapeutic efficacy against hematological malignancies in in vitro, ex vivo and in vivo pre-clinical studies. The TCL1-TG:p53 -/- mouse model exhibits a drug resistant phenotype resembling human CLL with unfavorable cytogenetic alterations and highly aggressive disease progression. AEB3103 greatly decreased the viability of TCL1-TG:p53 -/- cells cultured in vitro, whereas the CLL therapeutic, fludarabine, showed minimal cytotoxic effects. In vivo treatment of TCL1-TG:p53 -/- mice with AEB3103 resulted in an increase in median survival time (7 months, p<0.0001) compared to the untreated control group (3.5 months, p<0.001) and a fludarabine treated group (5.3 months, p<0.001). These results indicate a superior therapeutic effect of AEB3103 compared to fludarabine. Additionally, evaluation of AEB3103 in in vitro 2D cultures of patient-derived CLL and MM cells, and in ex vivo 3D cultures of cells derived from ALL and AML PDx models resulted in significant cell growth inhibition with therapeutically relevant IC50 values. Collectively these results demonstrate the sensitivity of hematological malignancies to modulation of GSH levels via AEB3103-mediated cyst(e)ine depletion. Disclosures Agnello: Aeglea BioTherapeutics: Employment. Alters:Aeglea BioTherapeutics: Employment, Equity Ownership. Tyler:Aeglea BioTherapeutics: Employment, Equity Ownership. Huang:Aeglea BioTherapeutics: Research Funding. Stone:Aeglea Biotherapeutics: Consultancy, Equity Ownership, Research Funding; University of Texas at Austin: Employment, Patents & Royalties: I am an inventor of technology related to this abstract. Georgiou:Aeglea Biotherapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Lowe:Aeglea BioTherapeutics: Employment, Equity Ownership. Rowlinson:Aeglea BioTherapeutics: Employment, Equity Ownership.

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Effects of tissue factor, thrombomodulin and elevated clotting factor levels on thrombin generation in the calibrated automated thrombogram

Thrombosis and Haemostasis ◽

10.1160/th09-03-0180 ◽

2009 ◽

Vol 102 (11) ◽

pp. 936-944 ◽

Cited By ~ 64

Author(s):

Kellie Machlus ◽

Emily Colby ◽

Jogin Wu ◽

Gary Koch ◽

Nigel Key ◽

...

Keyword(s):

Tissue Factor ◽

Thrombin Generation ◽

Relative Sensitivity ◽

Epidemiological Studies ◽

Peak Height ◽

Clotting Factor ◽

Time To Peak ◽

High Throughput Method ◽

Assay Conditions

SummaryElevated procoagulant levels have been correlated with increased thrombin generation in vitro and with increased venous thromboembolism (VTE) risk in epidemiological studies. hrombin generation tests are increasingly being employed as a high throughput method to provide a global measure of procoagulant activity in plasma samples. The objective of this study was to distinguish the effects of assay conditions [tissue factor (TF), thrombomodulin, platelets/lipids] and factor levels on thrombin generation parameters, and determine the conditions and parameters with the highest sensitivity and specificity for detecting elevated factor levels. Thrombin generation was measured using calibrated automated thrombography (CAT) in corn trypsin inhibitor (CTI)-treated platelet-free plasma (PFP) and plateletrich plasma (PRP). Statistical analysis was performed using logarithms of observed values with analysis of variance that accounted for experiment and treatment. he relative sensitivity of lag time (LT), time to peak (TTP), peak height and endogenous thrombin potential (ETP) to elevated factors XI, IX,VIII, X, and prothrombin was as follows: PFP initiated with 1 pM TF > PFP initiated with 5 pM TF > PRP initiated with 1 pM TF. For all conditions, inclusion of thrombomodulin prolonged the LT and decreased the peak and ETP; however, addition of thrombomodulin did not increase the ability of CAT to detect elevated levels of individual procoagulant factors. In conclusion, CAT conditions differentially affected the sensitivity of thrombin generation to elevated factor levels. Monitoring the peak height and/ or ETP following initiation of clotting in PFP with 1 pM TF was most likely to detect hypercoagulability due to increased procoagulant factor levels.

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Factor XI contributes to thrombin generation in the absence of factor XII

Blood ◽

10.1182/blood-2009-02-203604 ◽

2009 ◽

Vol 114 (2) ◽

pp. 452-458 ◽

Cited By ~ 96

Author(s):

Dmitri V. Kravtsov ◽

Anton Matafonov ◽

Erik I. Tucker ◽

Mao-fu Sun ◽

Peter N. Walsh ◽

...

Keyword(s):

Tissue Factor ◽

Thrombin Generation ◽

Factor Xa ◽

Factor Ix ◽

Factor Xii ◽

Factor Xi ◽

Factor Xii Deficiency ◽

Low Concentrations

Abstract During surface-initiated blood coagulation in vitro, activated factor XII (fXIIa) converts factor XI (fXI) to fXIa. Whereas fXI deficiency is associated with a hemorrhagic disorder, factor XII deficiency is not, suggesting that fXI can be activated by other mechanisms in vivo. Thrombin activates fXI, and several studies suggest that fXI promotes coagulation independent of fXII. However, a recent study failed to find evidence for fXII-independent activation of fXI in plasma. Using plasma in which fXII is either inhibited or absent, we show that fXI contributes to plasma thrombin generation when coagulation is initiated with low concentrations of tissue factor, factor Xa, or α-thrombin. The results could not be accounted for by fXIa contamination of the plasma systems. Replacing fXI with recombinant fXI that activates factor IX poorly, or fXI that is activated poorly by thrombin, reduced thrombin generation. An antibody that blocks fXIa activation of factor IX reduced thrombin generation; however, an antibody that specifically interferes with fXI activation by fXIIa did not. The results support a model in which fXI is activated by thrombin or another protease generated early in coagulation, with the resulting fXIa contributing to sustained thrombin generation through activation of factor IX.

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Ex Vivo Evaluation of the Effect of Plasma-Derived Factor VIII/Von Willebrand Factor in Patients with Severe Hemophilia_A on Prophylaxis with Emicizumab By Thrombin Generation Assay

Blood ◽

10.1182/blood-2021-147184 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4233-4233

Author(s):

Maria-Isabel Bravo ◽

Aida Raventós ◽

Alba Pérez ◽

Elena G Arias-Salgado ◽

María Teresa Alvarez Román ◽

...

Keyword(s):

Von Willebrand Factor ◽

Research Funding ◽

Thrombin Generation ◽

Ex Vivo ◽

Concomitant Treatment ◽

Healthy Controls ◽

Normal Range ◽

Von Willebrand ◽

Willebrand Factor

Abstract Introduction: Hemophilia A (HA) patients under emicizumab prophylaxis treatment may require the concomitant use of procoagulant factors for breakthrough bleedings or immune tolerance induction. Thromboembolic events have been described with the concomitant use of emicizumab and activated prothrombin complex concentrate (aPCC), but not with recombinant activated factor VII (rFVIIa). Previous studies showed that the in vitro combination of emicizumab and plasma-derived Factor VIII/Von Willebrand Factor (pdFVIII/VWF) had a non-additive effect on thrombin generation (TG)(Bravo M-I, et al J Thromb Haemost. 2020;18:1934-39). The aim of this study was to evaluate the TG resulting from ex vivo combination of plasma samples from HA patients treated with emicizumab, with a pdFVIII/VWF concentrate (Fanhdi ®, Grifols). Methods: Twelve adult patients with severe HA without inhibitors on prophylaxis with emicizumab and nine healthy controls were included in the study. Blood samples were drawn in citrate plus corn trypsin inhibitor tubes. Then, platelet poor plasma (PPP) was collected for the TG assay, which measures the whole kinetics of TG. Thrombin peak (TP) and endogenous thrombin potential (ETP) were calculated using calibrated automated thrombogram (Thrombinoscope ™ software, Stago) after in vitro activation of coagulation by trigger solution, PPP Reagent LOW TM (4 μM phospholipids/1 pM tissue factor), fluorogenic substrate and CaCl 2 (FLUKAkit TM) reagents (Diagnostica Stago). Fluorescence was read in a Fluoroskan Ascent reader (Thermo) equipped with a 390/460 filter set. Samples were spiked with increasing concentrations of pdFVIII/VWF (10 to 400 IU/dL), rFVIIa (0.9 µg/mL) or aPCC (0.5 U/mL). Results: TG from healthy control samples was measured to establish TP and ETP normal ranges. TP and ETP results obtained from HA plasma with emicizumab were lower than in healthy controls. The addition of pdFVIII/VWF as of 25 IU/kg (prophylaxis dose in HA w/o inhibitors) to samples from HA patients concomitantly treated with emicizumab restored TP and ETP levels within healthy controls normal range (Table 1). Increasing ex vivo concentrations of pdFVIII/VWF maintained TP and ETP similar to healthy controls. The highest concentration of concomitant treatment with pdFVIII/VWF (200 IU/kg) and emicizumab did not result in excessive TP and, importantly, ETP levels were always within the normal range. The combination with the bypassing agent rFVIIa moderately increased TP and ETP values up to normal range. However, when HA plasma was spiked with aPCC in the presence of emicizumab, TP and ETP dramatically increased above normal range resulting in a synergistic procoagulant profile. Conclusions: The concomitant use of pdFVIII/VWF in patients with prophylaxis with emicizumab did not trigger a multiplying effect on TG. These results were aligned with previous in vitro data and suggested the low risk of overdose and thrombotic events of concomitant treatment emicizumab with the pdFVIII/VWF concentrate in HA patients. Figure 1 Figure 1. Disclosures Bravo: Grifols: Current Employment, Other: Grifols is a manufacturer of the pdFVIII/VWF concentrate, Fanhdi®. Raventós: Grifols: Current Employment, Other: Grifols is a manufacturer of the pdFVIII/VWF concentrate, Fanhdi®. Pérez: Grifols: Current Employment, Other: Grifols is a manufacturer of the pdFVIII/VWF concentrate, Fanhdi®. Alvarez Román: Grifols: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Novo-Nordisk: Consultancy, Honoraria, Research Funding; Sobi: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; CSL-Behring: Consultancy, Honoraria, Research Funding; Biomarin: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding. Butta: CSL-Behring: Research Funding; Roche: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Novo-Nordisk: Speakers Bureau. Jiménez-Yuste: Bayer: Consultancy, Honoraria, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; BioMarin: Consultancy; Sobi: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; NovoNordisk: Consultancy, Honoraria, Research Funding; Grifols: Consultancy, Honoraria, Research Funding. Costa: Grifols: Current Employment, Other: Grifols is a manufacturer of the pdFVIII/VWF concentrate, Fanhdi®. Willis: Grifols: Current Employment, Other: Grifols is a manufacturer of the pdFVIII/VWF concentrate, Fanhdi®.

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Cord Blood Derived Natural Killer Cells Loaded with a Tetravalent Bispecific Antibody Construct (AFM13) As Off-the-Shelf Cell Therapy for CD30+ Malignancies

Blood ◽

10.1182/blood-2018-99-114954 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 341-341

Author(s):

Lucila Kerbauy ◽

Mecit Kaplan ◽

Pinaki P Banerjee ◽

Francesca Lorraine Wei Inng Lim ◽

Ana Karen Nunes Cortes ◽

...

Keyword(s):

T Cell ◽

Cord Blood ◽

Nk Cells ◽

Natural Killer ◽

Research Funding ◽

Bispecific Antibody ◽

Nk Cell ◽

Ex Vivo

Abstract Chimeric antigen receptors to redirect T cell specificity against tumor antigens have shown remarkable clinical responses against CD19+ malignancies. However, the manufacture of an engineered autologous T cell product is expensive and cumbersome. Natural killer (NK) cells provide an alternative source of immune effectors for the treatment of cancer. NK cell cytolytic function can be directed towards specific targets by exploiting their ability to mediate antibody-dependent cellular cytotoxicity (ADCC) through the NK cell Fc receptor, CD16 (FcγRIIIa). AFM13 is a tetravalent bispecific antibody construct based on Affimed's ROCK™ platform. AFM13 is bispecific for CD30 and CD16A, designed for the treatment of CD30 expressing malignancies. It binds CD16A on the surface of NK cells, thus activating and recruiting them to CD30 expressing tumor cells and mediating subsequent tumor cell killing. Since autologous NK effector function is impaired in many patients with malignancies, we propose to overcome this by the use of allogeneic NK cells in combination with AFM13. Cord blood (CB) is a readily available ("off-the-shelf") source of allogeneic NK cells that can be expanded to large, highly functional therapeutic doses. The feasibility and safety of therapy with allogeneic ex vivo expanded CB-derived NK cells have been shown by our group and others. In this study, we hypothesized that we can redirect the specificity of NK cells against CD30+ malignancies by preloading ex vivo activated and expanded CB-derived NK cells with AFM13 prior to adoptive infusion. Briefly, mononuclear cells were isolated from fresh or frozen CB units by ficoll density gradient centrifugation. CD56+ NK cells were cultured with rhIL-12, rhIL-18 and rhIL-15 for 16 hrs, followed by ex vivo expansion with rhIL-2 and irradiated (100 Gy) K562-based feeder cells expressing membrane-bound IL-21 and CD137-ligand (2:1 feeder cell:NK ratio). After 14 days, NK cells were loaded with serial dilutions of AFM13 (0.1, 1, 10 and 100 mg/ml). After washing twice with PBS, we tested the effector function of AFM13-loaded NK-cells (AFM13-NK) compared to expanded CB-NK cells without AFM13 against Karpas-299 (CD30 positive) and Daudi (CD30 negative) lymphoma cell lines by 51Cr release and intracellular cytokine production assays. AFM13-NK cells killed Karpas-299 cells more effectively at all effector:target ratios tested than unloaded NK cells (Figure 1) and produced statistically more INFγ and CD107a (P=0.0034; P=0.0031 respectively, n=4). In contrast, AFM13-NK cells and unloaded NK cells exerted similar cytotoxicity against Daudi cells. Next, we established the optimal concentration of AFM13 for loading (determined to be 100 μg/ml) and the optimal incubation time to obtain maximal activity (1 h) in a series of in vitro experiments. We also confirmed that the activity of AFM13-NK cells against Karpas-299 cells remains stable for at least 72h post-wash (Figure 2). Additionally, we characterized the phenotype of AFM13-NK vs. unloaded NK cells by flow cytometry using monoclonal antibodies against 22 markers, including markers of activation, inhibitory receptors, exhaustion markers and transcription factors. Compared to unloaded NK cells, AFM13-NK cells expressed higher levels of CD25, CD69, TRAIL, NKp44, granzyme B and CD57, consistent with an activated phenotype. We next tested the in vivo anti-tumor efficacy of AFM13-NK cells in an immunodeficient mouse model of FFluc-Karpas-299. Briefly, six groups of NOD/SCID/IL2Rγc null mice (n=5 per group) were transplanted by tail-vein injection with 1 x 10e5 FFluc-transduced Karpas cells. Group 1 and 6 received tumor alone or tumor + AFM13 and served as a control. Groups 2-4 receive Karpas FFLuc with either expanded NK cells or AFM13-NK cells (NK cells loaded with AFM13) or expanded NK cells and AFM13 injected separately. Group 5 received AFM13-NK cells without tumor. Initial studies confirm the antitumor activity of AFM13-NK cells. In summary, we have developed a novel premixed product, comprised of expanded CB-NK cells loaded with AFM13 to 'redirect' their specificity against CD30+ malignancies. The encouraging in vitro and in vivo data observed in this study, provide a strong rationale for a clinical trial to test the strategy of an off-the-shelf adoptive immunotherapy with AFM13-loaded CB-NK cells in patients with relapsed/refractory CD30+ malignancies. Disclosures Champlin: Sanofi: Research Funding; Otsuka: Research Funding. Koch:Affimed GmbH: Employment. Treder:Affimed GmbH: Employment. Shpall:Affirmed GmbH: Research Funding. Rezvani:Affirmed GmbH: Research Funding.

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Evaluating the Antitumor Activity of MLN9708 in a Disseminated Mouse Model of Double Transgenic iMyc Ca/Bcl-XL Plasma Cell Malignancy.

Blood ◽

10.1182/blood.v114.22.3835.3835 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3835-3835 ◽

Cited By ~ 1

Author(s):

Michael Fitzgerald ◽

Yueying Cao ◽

Bret Bannerman ◽

Zhi Li ◽

Olga Tayber ◽

...

Keyword(s):

Antitumor Activity ◽

Mouse Model ◽

Lymph Nodes ◽

Plasma Cell ◽

Research Funding ◽

Ex Vivo ◽

Employment Equity ◽

Equity Ownership

Abstract Abstract 3835 Poster Board III-771 Introduction The first generation proteasome inhibitor VELCADE® (bortezomib) is indicated for the treatment of patients with multiple myeloma (MM), a form of plasma cell malignancy (PCM). MLN9708 is our novel proteasome inhibitor that selectively and reversibly binds to, and potently inhibits the b5 site of the 20s proteasome in preclinical studies. We have recently demonstrated that MLN9708 significantly prolongs tumor-free survival of double transgenic iMycCa/Bcl-XL mice, a genetically-engineered mouse model of de novo PCM. Here we describe the in vivo evaluation of cell lines derived from double transgenic iMycCa/Bcl-XL mice and the antitumor activity of MLN9708 in a disseminated mouse model of iMycCa/Bcl-XL PCM. Materials MLN9708 immediately hydrolyzes to MLN2238, the biologically active form, upon exposure to aqueous solutions or plasma. MLN2238 was used for all preclinical studies described below. Double transgenic iMycCa/Bcl-XL mice develop de novo PCM, in which neoplastic plasma cell development is driven by the targeted expression of the oncoprotein Myc and anti-apoptotic Bcl-XL (J. Clin. Invest. 113:1763-1773, 2004). DP54 and DP42 are plasma cell tumor cell lines isolated from the bone marrow and lymph nodes, respectively, of syngeneic mice previously inoculated with iMycCa/Bcl-XL tumors (Cancer Res. 67:4069-4078, 2007). In vitro, DP54 and DP42 cells express both the Myc and Bcl-XL transgenes, various plasma cell and B-cell markers including CD38, CD138 and B220, and have gene expression profiles very similar to human MM. Methods Cell viability studies were performed to determine the antiproliferative effects of MLN2238 in DP54 and DP42 cells in vitro. To evaluate DP54 and DP42 cells in vivo, these cells were aseptically inoculated into the tail vein of NOD-SCID mice. Progressions of the resultant PCM were monitored and tumor burdens were evaluated by magnetic resonance imaging (MRI), ex vivo mCT imaging, and histopathology. Mouse plasma samples were collected at the end of the studies and levels of immunoglobulin were assessed. To establish a preclinical disseminated mouse model of iMycCa/Bcl-XL PCM, freshly dissociated DP54-Luc cells (constitutively expressing firefly luciferase under a mouse Ig-k promoter) were aseptically inoculated into the tail vein of NOD-SCID mice. Once tumor growth has been established, mice were randomized into treatment groups and then treated with vehicle, bortezomib (at 0.7mg/kg intravenously [IV] twice weekly [BIW]) or MLN2238 (at 11 mg/kg IV BIW) for 3 consecutive weeks. Tumor burden was measured by bioluminescent imaging. Results In vitro, both DP54 and DP42 cells were sensitive to MLN2238 treatment (LD50 values of 14 and 25 nM, respectively). In vivo, NOD-SCID mice rapidly succumbed to PCM after being inoculated with DP54 and DP42 cells (25 and 14 days post-inoculation, respectively), where the disease was accompanied by marked elevation of plasma immunoglobulins. MRI scans revealed the presence of multiple lesions and several abnormalities were found including: cranial deformation, bowel distortion, splenomegaly and renal edema. Tumor infiltrates, ranging from minor to extensive, were identified in multiple organ compartments (brain<kidney<liver<lymph nodes<spleen<bone marrow) by histopathological analysis. Ex vivo mCT imaging has also revealed signs of bone erosion in the cranial sagittal sutures. Dissemination of DP54-Luc cells after tail vein inoculations was detected by in vivo bioluminescent and confirmed by ex vivo imaging where luminescent tumor nodules were identified in the spleen, kidneys, liver, intestine, lymph nodes, spinal bone and cranium. To assess the antitumor activity of MLN2238, an efficacy study was performed using the DP54-Luc disseminated model. Tumor burden (bioluminescence), skeletal malformation (mCT) and overall survival after treatment with bortezomib and MLN2238 will be presented. Conclusion The DP54-Luc disseminated mouse model of double transgenic iMycCa/Bcl-XL PCM recapitulated several key features of human MM and provided real-time assessment of novel MM therapy preclinically. MLN9708 is currently in human clinical development for both hematological and solid tumor indications. Disclosures: Cao: Milllennium: Employment, Equity Ownership. Bannerman:Milllennium: Employment. Li:Milllennium: Employment. Bradley:Milllennium: Employment, Equity Ownership, Research Funding. Silverman:Milllennium: Employment. Janz:Milllennium: Research Funding. Van Ness:Milllennium: Research Funding. Kupperman:Milllennium: Employment. Manfredi:Milllennium: Employment. Lee:Milllennium: Employment, Equity Ownership.

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Afibrinogenemia: a Disorder of Paradoxical Bleeding and Clotting.

Blood ◽

10.1182/blood.v114.22.3477.3477 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3477-3477

Author(s):

Mindy L. Grunzke ◽

Chris Bombardier ◽

Linda J. Jacobson ◽

Neil A. Goldenberg ◽

Marilyn J. Manco-Johnson

Keyword(s):

Research Funding ◽

Thrombin Generation ◽

Dose Dependence ◽

Fibrinogen Concentrate ◽

Bleeding Events ◽

Time To Peak ◽

Post Infusion ◽

Life Threatening ◽

Thrombotic Complications ◽

Peripheral Arterial

Abstract Abstract 3477 Poster Board III-414 Background Congenital afibrinogenemia (CA) is a rare bleeding disorder. While thrombosis is a recognized complication of dysfibrinogemia, the risk for thrombosis in CA has not been well studied. Life-threatening thrombosis with onset shortly after puberty has developed in 2 of 3 patients with CA followed through the coagulation program at The Children's Hospital, Colorado. Patient 1 suffered recurrent peripheral arterial thrombosis following cryoprecipitate administration during adolescence, massive myocardial infarction (MI) at age 19 years and death from spontaneous intracranial hemorrhage at age 20 years. Patient 2 has had superficial thrombophlebitis following intravenous (IV) infusion of fibrinogen concentrate, as well as bilateral pulmonary embolism with infarction 1 month following last infusion, without further recurrence over the course of 6 months of judicious use of anticoagulation and fibrinogen replacement. Patient 3 is 14 years old and receives fibrinogen replacement episodically for bleeding events, without thrombotic complications to date. Objective The object of this study was to investigate overall thrombin and plasmin generation potential and overall coagulative and fibrinolytic potential in CA, in order to generate hypotheses regarding pathophysiology of bleeding and thrombotic complications. Methods Plasma was collected from 2 CA patients at asymptomatic baseline states and following fibrinogen concentrate replacement. Plasma was isolated from whole blood within 1 hour, via double centrifugation at 2500 x g at 4°C x 15 minutes, and stored at -70°C until time of assay. Standard assays included fibrinogen activity by Clauss clotting assay and thrombin-antithrombin complexes (TAT) by ELISA (Siemens, Marburg, GE). Measurement of overall coagulative and fibrinolytic capacity in plasma was performed by Clot Formation and Lysis (CloFAL) global assay, as previously described (Goldenberg et al., Haemophilia, 2008). Simultaneous Thrombin and Plasmin generation assay (STP) was performed by fluorometric method with the same reagents as used in the CloFAL assay (dilute tissue factor, phospholipid, tissue plasminogen activator), as previously reported (Grunzke et al., J Thromb Haemost 2009 (abstract)). Results TAT was elevated to twice the upper limit of normal (9.4 mcg/L) in patient 1 six months following MI. In patient 2, TAT increased progressively from age 15 (2.9 mcg/L) to 20 years (13.7 mcg/L) while STP showed progressive shortening of time to peak thrombin generation by 60% over these 5 years (Figure 1). Baseline plasmin generation was nearly undetectable in both patients studied. As shown in Table I, TAT increased significantly at peak post infusion of fibrinogen concentrate in both patients evaluated during half-life and recovery studies. As shown in Figure 2, panels A and B, coagulative capacity by CloFAL assay and velocity of plasmin generation by STP showed dose dependence to fibrinogen following IV replacement with concentrate in patient 3. Discussion These data suggest that baseline and post infusion thrombin generation may be increased in post pubertal patients with CA. Furthermore, given undetectable plasmin generation in the absence of fibrinogen, the hemostatic balance in CA is simultaneously hemorrhagic and prothrombotic. These preliminary findings call for further prospective evaluation in other identified patients with CA. Disclosures: Grunzke: National Hemophilia Foundation/Baxter Clinical Fellowship Award: Research Funding. Manco-Johnson:CSL Behring: Honoraria, Research Funding.

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